Methods of using zscan4 for rejuvenating human cells

ABSTRACT

The present disclosure relates to methods for increasing telomere length in one or more human cells and/or increasing genome stability of one or more human cells, for example by contacting one or more human cells with an agent that increases expression of Zscan4 in the one or more human cells. Methods of treating a subject in need of telomere lengthening, treating a disease or condition associated with a genomic and/or chromosome abnormality, of rejuvenating one or more human cells, of rejuvenating tissues or organs, and of rejuvenating a subject in need thereof, for example by contacting one or more human cells in the subject with an agent that increases expression of Zscan4, or by administering to a subject in need thereof, an agent that increases expression of Zscan4 are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/427,038, filed on May 30, 2019, which is a divisional of U.S. patentapplication Ser. No. 14/776,707, with an international filing date ofMar. 14, 2014, which is a National Stage of International PatentApplication No. PCT/US2014/029537, filed Mar. 14, 2014, which claims thebenefit of U.S. Provisional Patent Application No. 61/800,668, filedMar. 15, 2013, the disclosures of which are herein incorporated byreference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 699442000802SEQLIST.TXT,date recorded: Jun. 30, 2022, size: 106,660 bytes).

FIELD

The present disclosure relates to methods for increasing telomere lengthin one or more human cells and/or increasing genome stability of one ormore human cells, for example by contacting one or more human cells withan agent that increases expression of Zscan4 in the one or more humancells. The present disclosure further provides methods of treating asubject in need of telomere lengthening, treating a disease or conditionassociated with a genomic and/or chromosome abnormality, of rejuvenatingone or more human cells, of rejuvenating tissues or organs, and ofrejuvenating a subject in need thereof, for example by contacting one ormore human cells in the subject with an agent that increases expressionof Zscan4, or by administering to a subject in need thereof, an agentthat increases expression of Zscan4.

BACKGROUND

Telomeres are repetitive DNA sequences accompanied by proteins that capand protect the end of each chromosome from continuous degradation ineach cell cycle, thereby securing and protecting chromosomal integrity.Telomere shortening may also lead to cancer by contributing to genomicinstability (Raynaud et al., Crit. Rev Oncol Hematol 66:99-117, 2008),and has been associated with aging and cellular senescence (Yang,Cytogenet Genome Res 122:211-218, 2008). It is well established thattelomeres get gradually shorter during the course of normal aging. Ithas been reported that up to 200 base pairs of telomere DNA are lostwith each round of DNA replication. For example, in new-born humans,peripheral blood lymphocytes have approximately 10 kb of telomere DNA atboth ends of each chromosome, which gradually shorten to approximately 6kb by the age of 70. It is also known that environmental factors andlife-style factors can accelerate telomere shortening. It is believedthat such telomere shorting is associated with age-related cellulardecline. It is also believed that telomere shortening limits the numberof cell divisions, which ultimately results in limiting human life span.It is also known that humans are born with differing lengths oftelomeres. For example, some humans start with approximately 8 kb oftelomeres, while others start with approximately 12 kb of telomeres.Accordingly, humans with shorter telomeres may be more susceptible todeveloping certain age-related pathological conditions at an earlier agethan those with longer telomeres. Such pathological conditions include,for example, immunological deficiencies, chronic ulcers,atherosclerosis, age-related blindness due to a proliferative decline ofretinal pigmented epithelial cells, and cancer.

Moreover, there are various diseases and disorders that are alsoassociated with telomere shortening (Armanios and Blackburn, Nat RevGenet. 2012 October; 13(10):693-704). Examples of genetic diseases thatcan cause telomere shortening include dyskeratosis congenita,Hoyeraal-Hreidarsson syndrome, Revesz syndrome, and Coats plus syndrome.Additionally, it was recently shown that a significant fraction ofidiopathic pulmonary fibrosis (IPF) is caused by telomere shortening.Similarly, some liver cirrhosis and pancreatic fibrosis may be caused bytelomere shortening. Considering the prevalence of such pathologicalconditions, it appears that diseases caused by telomere shortening aremore common than previously thought.

Another example of a disease associated with telomere shortening isFanconi anemia. Fanconi anemia is a rare autosomal recessive disease.Fanconi anemia is an inherited bone marrow failure syndrome that ischaracterized by progressive pancytopenia and cancer susceptibility(Bogliolo et al., Mutagenesis. 2002 November; 17(6):529-38). It has beenreported that Fanconi anemia patients show accelerated telomereshortening (Leteurte et al., Br. J. Haematol., 1999; Ball et al., Blood,1998; Hanson et al., Cytogenet. Cell Genet. 2001; and Callen, et al.,Hum Mol Genet. 2002 Feb. 15; 11(4):439-44).

One potential method of treating these various telomereshortening-associated diseases and disorders is to use telomerase tolengthen the shortened telomeres. Telomerase has been identified as themajor enzyme known to be involved in telomere elongation maintenance.While telomerase is active in embryonic stem cells, telomerase isusually not expressed in non-embryonic (i.e., adult cells), such assomatic cells. Thus the reactivation of telomerase or forced expressionof telomerase in adult cells may be used to increase telomere length.However, one potential problem with the use of telomerase is that thecontinuous expression of telomerase is often associated withtumorigenesis and cancerous transformation. Accordingly, expression oftelomerase is not an ideal way to increase telomere length in patientssuffering from diseases or conditions associated with telomereshortening.

Another potential method to lengthen telomeres is to use a recentlydiscovered component of a Chinese herb (TA-65) that can potentiallyincrease telomere length (Harley et al., Rejuvenation Research 14:45-56,2011). However, it has not been well established that this herb caneffectively lengthen telomeres. Moreover, use of this herb would requirelong-term continuous administration of drugs to treat patients in needof telomere lengthening.

Additionally, it has recently been shown that Zscan4 (Zinc finger andscan domain-containing protein 4) is required for the maintenance ofgenome stability and normal karyotype in mouse embryonic stem cells andis expressed in mouse embryos and embryonic stem cells (Falco et al.,Dev Biol 307:539-550, 2007; Zalzman et al., Nature 464:858-863, 2010;PCT Publication Nos. WO 2008/118957, WO 2011/02880, WO 2012/103235, WO2012/129342, WO 2012/158561, and WO 2012158564; and U.S. PatentApplication Publication Nos. US 2010/0105043, US 2012/0129161, and US2012/0156305). It has also been shown that Zscan4 expression in mouseembryonic stem cells is associated with telomere elongation (Zalzman etal., Nature 464:858-863, 2010; PCT Publication Nos. WO 2011/02880, WO2012/129342, and WO 2012158564; and U.S. Patent Application PublicationNo. US 2012/0156305). While, it has been shown that the human genomealso contains a ZSCAN4 gene, none of Falco et al., Dev Biol 307:539-550,2007; Zalzman et al., Nature 464:858-863, 2010; PCT Publication Nos. WO2008/118957, WO 2011/02880, WO 2012/103235, WO 2012/129342, WO2012/158561, and WO 2012158564; or U.S. Patent Application PublicationNos. US 2010/0105043, US 2012/0129161, and US 2012/0156305 provideexperimental support demonstrating that Zscan4 expression leads to sameeffects in human cells as it does in mouse embryonic cells. It isparticularly unclear whether human ZSCAN4 would have the same functionas mouse Zscan4, as the mouse genome contains six Zscan4 genes and threeZscan4 pseudogenes while the human genome only contains one ZSCAN4 gene(PCT Publication No. WO 2008/118957). Moreover, it is unknown whetherZSCAN4 expression in human cells, such as somatic cells involved indiseases and conditions associated with telomere shortening, would havethe same effect as shown for mouse embryonic stem cells.

SUMMARY

Accordingly, there exists a need for improved approaches for increasingtelomere length and correcting genomic and/or chromosome abnormalitiesin human cells in order to treat diseases or conditions associated withtelomere shortening and genomic abnormalities.

In order to meet the above need, the present disclosure provides novelmethods of increasing telomere length, increasing chromosome and/orgenome stability in human cells, correcting chromosome and/or karyotypeabnormalities (e.g., trisomy 21), in human cells, and/or rejuvenatinghuman cells, by contacting the human cells with an agent that increasesexpression of Zscan4 (Zinc finger and scan domain-containing protein 4)in the cells. As used herein, the term “Zscan4” refers to Zscan4polypeptides and polynucleotides, such as genes, encoding Zscan4polypeptides from any species, including mouse and human. As usedherein, the term “ZSCAN4” refers specifically to human Zscan4polypeptides and polynucleotides, such as genes, encoding Zscan4polypeptides.

The present disclosure also provides novel methods of treating a diseaseor condition associate with a telomere, chromosome and/or karyotypeabnormality, increasing genomic stability and correcting karyotypeabnormalities in human oocyte cells, human fertilized oocytes, and humanpreimplantation embryos, rejuvenating a tissue or organ, and/orrejuvenating a subject in need thereof, by administering to a subject inneed thereof an agent that increases expression of Zscan4. In someembodiments, the human cells are human adult cells (i.e., non-embryoniccells).

Moreover, the present disclosure is based, at least in part, on thesurprising discovery that Zscan4 expression in human cells, such asfibroblast cells, rapidly and dramatically increases the length oftelomeres in the cells after just two days. In particular, as disclosedin the Example 8 below, Zscan4 expression in human fibroblast resultedin about a 40% increase in telomere length within three days.Additionally, expression of Zscan4 in human fibroblasts isolated from apatient with Fanconi anemia resulted in about a 160% increase intelomere length within three days. Surprisingly, Zscan4 expression in apopulation of fibroblast cells isolated from a Down syndrome patient wasalso able to dramatically reduce the percentage of cells in thepopulation with trisomy 21. In particular, as disclosed in Example 15below, Zscan4 expression in a population of fibroblast cells isolatedfrom a Down syndrome patient was able to correct the trisomy 21abnormality in approximately 55% of the cells.

The results disclosed herein are particularly surprising given that itis believed that it has never before been shown that Zscan4 expressioncan increase telomere length in human cells. The results disclosedherein are also unexpected. While Zscan4 expression has been previouslyshown to increase telomere length in mouse embryonic stem (ES) cells,the differences not only between human ZSCAN4 and mouse Zscan4, but alsobetween the biology of human cells and mouse cells, as well as betweenES cells and non-ES cells such as adult cells, would not lead one toexpect that Zscan4 expression in human cells would also increasetelomere length. This is particularly relevant given that it has beenpreviously demonstrated that transcriptional regulatory elements in thehuman and mouse genomes differ dramatically. This is exceptionallystriking when considering that even transcription factors with conservedfunction in both human and mouse exhibit a significant degree ofspecies-specific binding event preferences (Odom et al., Nature Genetics6:39, 2007).

Advantageously, utilizing agents that increase Zscan4 expression, suchas nucleic acid molecules encoding Zscan4, can be used to increase thesuccess rate of in vitro fertilization (IVF) and successful pregnanciesin older women by rejuvenating and/or correcting genomic and/orchromosomal abnormalities, such as aneuploidy, in oocyte cells,fertilized oocytes, or preimplantation embryos. Additionally, utilizingagents that increase Zscan4 expression, such as nucleic acid moleculesencoding Zscan4, can be used to treat a patient suffering from a diseaseor condition associated with telomere shortening, such as Fanconianemia, by increasing the length of telomeres in cells of the patientaffected by the disease or condition. Furthermore, agents that increaseZscan4 expression, such as nucleic acid molecules encoding Zscan4, canbe used to also rejuvenate cells in an individual, tissues in anindividual, or organs in an individual; or to rejuvenate individuals byincreasing the length of telomeres in aged cells, tissues, organs andindividuals caused by telomere shortening.

Accordingly, certain aspects of the present disclosure relate to amethod of increasing telomere length in one or more human cells, bycontacting the one or more human cells with an agent that increasesexpression of Zscan4 in the human cell, where increased expression ofZscan4 induces telomere lengthening in the one or more human cells ascompared to one or more corresponding human cells that are not contactedwith the agent.

Other aspects of the present disclosure relate to a method of treating asubject in need of telomere lengthening, by contacting one or more humancells in the subject with an agent that increases expression of Zscan4in the one or more human cells, where increased expression of Zscan4induces telomere lengthening in the one or more human cells.

Other aspects of the present disclosure relate to a method of treating asubject in need of telomere lengthening, by: i. isolating one or morehuman cells in need of telomere lengthening from the subject; ii.contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where increasingexpression of Zscan4 induces telomere lengthening in the one or morehuman cells; and iii. administering the contacted one or more humancells to the subject.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a telomere abnormality, byadministering to a subject in need thereof an agent that increasesexpression of Zscan4 in one or more human cells in the subject, whereincreasing expression of Zscan4 induces telomere lengthening in the oneor more human cells to treat the disease or condition associated with atelomere abnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a telomere abnormality, by: i.isolating one or more human cells from a subject suffering from adisease or condition associated with a telomere abnormality; ii.contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where increasingexpression of Zscan4 induces telomere lengthening in the one or morehuman cells; and iii. administering the contacted one or more humancells to the subject to treat the disease or condition associated with atelomere abnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a chromosome abnormality, byadministering to a subject in need thereof an agent that increasesexpression of Zscan4 in one or more human cells in the subject, whereincreasing expression of Zscan4 induces correction of the chromosomeabnormality in the one or more human cells to treat the disease orcondition associated with a chromosome abnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a chromosome abnormality, by: i.isolating one or more human cells from a subject suffering from adisease or condition associated with a chromosome abnormality; ii.contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where increasingexpression of Zscan4 induces correction of the chromosome abnormality inthe one or more human cells; and iii. administering the contacted one ormore human cells to the subject to treat the disease or conditionassociated with a chromosome abnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a karyotype abnormality, byadministering to a subject in need thereof an agent that increasesexpression of Zscan4 in one or more human cells in the subject, whereincreasing expression of Zscan4 induces correction of the karyotypeabnormality in the one or more human cells to treat the disease orcondition associated with a karyotype abnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a karyotype abnormality, by: i.isolating one or more human cells from a subject suffering from adisease or condition associated with a karyotype abnormality; ii.contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where increasingexpression of Zscan4 induces correction of the karyotype abnormality inthe one or more human cells; and iii. administering the contacted one ormore human cells to the subject to treat the disease or conditionassociated with a karyotype abnormality.

In some embodiments that may be combined with any of the precedingembodiments, the karyotype abnormality is selected from a chromosomenullisomy, a chromosome monosomy, a chromosome trisomy, a chromosometetrasomy, and a chromosome pentasomy. In some embodiments that may becombined with any of the preceding embodiments, the karyotypeabnormality is selected from trisomy 21, trisomy 16, trisomy 18, trisomy13, monosomy X, XXX aneuploidy, XXY aneuploidy, XYY aneuploidy, and 1p36duplication. In some embodiments that may be combined with any of thepreceding embodiments, the disease or condition associated with akaryotype abnormality is selected from dup(17)(p11.2p11.2) syndrome,Pelizaeus-Merzbacher disease, dup(22)(q11.2q11.2) syndrome, cat-eyesyndrome, Cri-du-chat syndrome, Wolf-Hirschhorn, Williams-Beurensyndrome, Charcot-Marie-Tooth disease, Hereditary neuropathy withliability to pressure palsies, Smith-Magenis syndrome,Neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome,DiGeorge syndrome, Steroid sulfatase deficiency, Kallmann syndrome,Microphthalmia with linear skin defects, Adrenal hypoplasia, Glycerolkinase deficiency, Pelizaeus-Merzbacher disease, Testis-determiningfactor on Y, Azoospermia (factor a), Azoospermia (factor b), Azoospermia(factor c), and 1p36 deletion. In some embodiments that may be combinedwith any of the preceding embodiments, the disease or condition is oneor more diseases or conditions selected from diseases of telomereshortening, bone marrow failure syndromes, age-related telomereshortening diseases or disorders, and premature aging diseases ordisorders. In some embodiments that may be combined with any of thepreceding embodiments, the disease or condition is a disease of telomereshortening selected from dyskeratosis congenita, Hoyeraal-Hreidars sonsyndrome, Revesz syndrome, Coats plus syndrome, idiopathic pulmonaryfibrosis, liver cirrhosis, pancreatic fibrosis, Alzheimer's disease, andosteoarthritis. In some embodiments that may be combined with any of thepreceding embodiments, the disease or condition is a bone marrow failuresyndrome selected from Fanconi anemia, amegakaryocytic thrombocytopenia,aplastic anemia, Diamond Blackfan anemia, dyskeratosis congenita,paroxysmal nocturnal hemoglobinuria (PNH), Pearson syndrome, ShwachmanDiamond syndrome, thrombocytopenia, and myelodysplastic syndrome. Insome embodiments that may be combined with any of the precedingembodiments, the disease or condition is an age-related telomereshortening disease or disorder, a premature aging disease or disorder,or both selected from Werner syndrome, Bloom's syndrome,Hutchinson-Gilford progeria syndrome, Cockayne syndrome, Xerodermapigmentosa, Ataxia telangiectasia, Rothmund Thomson syndrome,Trichothiodystrophy, Juberg-Marsidi syndrome, and Down syndrome. In someembodiments that may be combined with any of the preceding embodiments,the disease or condition is one or more diseases or conditions selectedfrom immunological deficiencies, an autoimmune disease, an autoimmunedisorder, chronic ulcers, atherosclerosis, cancer, a neurologic injury,a degenerative disorder, a neurodegenerative disorder, wound healing,muscle repair, cardiac muscle repair, cartilage replacement, arthritis,osteoarthritis, tooth regeneration, blindness, age-related blindness dueto proliferative decline of retinal pigmented epithelial cells,deafness, bone marrow failure, bone marrow transplant, diabetes,muscular dystrophy, Duchenne muscular dystrophy, a genetic disease, agenetic mutation, and DNA damage. In some embodiments that may becombined with any of the preceding embodiments, the disease or conditionis a cancer selected from cancers of the heart (e.g., angiosarcoma,fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma,fibroma, lipoma and teratoma), lung cancers (e.g., bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma);gastrointestinal tract cancers (e.g., esophagus (squamous cellcarcinoma, adenocarcinoma, leiomyosarcoma, lymphoma); stomach cancers(carcinoma, lymphoma, leiomyosarcoma); pancreatic cancers (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma); small bowel cancers (adenocarcinoma, lymphoma, carcinoidtumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma); large bowel cancers (adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma); genitourinary tract cancers (e.g.,kidney (adenocarcinoma, Wilms' tumor, nephroblastoma, lymphoma,leukemia); bladder and urethra cancers (squamous cell carcinoma,transitional cell carcinoma, adenocarcinoma); prostate cancers(adenocarcinoma, sarcoma); testis cancers (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); livercancers (e.g., hepatoma (hepatocellular carcinoma), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma); bonecancers (e.g., osteogenic sarcoma (osteosarcoma), fibrosarcoma,malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma,malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignantgiant cell tumor, chordoma, osteochondroma (osteocartilaginousexostoses), benign chondroma, chondroblastoma, chondromyxofibroma,osteoid osteoma and giant cell tumors); nervous system cancers (e.g.,skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans),meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma, pinealoma, glioblastomamultiforme, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma));gynecological cancers (e.g., uterus (endometrial carcinoma), cervix(cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovariancarcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma,endometrioid tumors, Brenner tumor, clear cell carcinoma, unclassifiedcarcinoma, granulosa-theca cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma,embryonal rhabdomyosarcoma, fallopian tubes (carcinoma)); hematologiccancers (e.g., blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma)); skin cancers(e.g., malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Kaposi's sarcoma, moles, dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis); and adrenal gland cancers (e.g.,neuroblastoma). In some embodiments that may be combined with any of thepreceding embodiments, the disease or condition is an autoimmune diseaseselected from thyroiditis, Goodpasture's disease, rheumatoid arthritis,juvenile oligoarthritis, collagen-induced arthritis, adjuvant-inducedarthritis, Sjogren's syndrome, multiple sclerosis, experimentalautoimmune encephalomyelitis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, autoimmune gastric atrophy, pemphigusvulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes,myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosingcholangitis, sclerosing sialadenitis, systemic lupus erythematosis,autoimmune thrombocytopenia purpura, Addison's disease, systemicsclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia,and pernicious anemia. In some embodiments that may be combined with anyof the preceding embodiments, the disease or condition is aneurodegenerative disease selected from adrenoleukodystrophy (ALD),alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease,amyotrophic lateral sclerosis, Lou Gehrig's Disease, ataxiatelangiectasia, Batten disease, Spielmeyer-Vogt-Sjogren-Batten disease,bovine spongiform encephalopathy (BSE), Canavan disease, cerebral palsy,Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,familial fatal insomnia, frontotemporal lobar degeneration, Huntington'sdisease, HIV-associated dementia, Kennedy's disease, Krabbe's disease,Lewy body dementia, neuroborreliosis, Machado-Joseph disease,Spinocerebellar ataxia type 3, Multiple System Atrophy, multiplesclerosis, narcolepsy, Niemann Pick disease, Parkinson's disease,Pelizaeus-Merzbacher Disease, Pick's disease, primary lateral sclerosis,prion diseases, progressive supranuclear palsy, Refsum's disease,Sandhoff disease, Schilder's disease, subacute combined degeneration ofspinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease, Batten disease, spinocerebellarataxia, spinal muscular atrophy, Steele-Richardson-Olszewski disease,Tabes dorsalis, and toxic encephalopathy.

Other aspects of the present disclosure relate to a method of treating acancer, by administering to a subject in need thereof an agent thatincreases expression of Zscan4 in one or more cancer cells in thesubject, where increasing expression of Zscan4 represses growth of theone or more cancer cells, thereby treating the cancer. Other aspects ofthe present disclosure relate to a method of improving responsiveness tochemotherapy in a cancer patient, by administering to a subject in needthereof an agent that reduces expression of endogenous ZSCAN4 in one ormore cancer stem cells in the subject, where reducing expression ofendogenous ZSCAN4 reduces or eliminates resistance to one or morechemotherapeutic agents in the one or more cancer stem cells, therebyimproving responsiveness to the one or more chemotherapeutic agents inthe subject. In some embodiments, the agent that reduces expression ofendogenous ZSCAN4 is an siRNA or shRNA specific for ZSCAN4. In someembodiments, the cancer selected from cancers of the heart (e.g.angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma,rhabdomyoma, fibroma, lipoma and teratoma), lung cancers (e.g.,bronchogenic carcinoma (squamous cell, undifferentiated small cell,undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma); gastrointestinal tract cancers (e.g.,esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma,lymphoma); stomach cancers (carcinoma, lymphoma, leiomyosarcoma);pancreatic cancers (ductal adenocarcinoma, insulinoma, glucagonoma,gastrinoma, carcinoid tumors, vipoma); small bowel cancers(adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma); large bowelcancers (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); genitourinary tract cancers (e.g., kidney (adenocarcinoma,Wilms' tumor, nephroblastoma, lymphoma, leukemia); bladder and urethracancers (squamous cell carcinoma, transitional cell carcinoma,adenocarcinoma); prostate cancers (adenocarcinoma, sarcoma); testiscancers (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma); liver cancers (e.g., hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma); bone cancers (e.g.,osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor,chordoma, osteochondroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors); nervous system cancers (e.g., skull (osteoma,hemangioma, granuloma, xanthoma, osteitis deformans), meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma, pinealoma, glioblastomamultiforme, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma));gynecological cancers (e.g., uterus (endometrial carcinoma), cervix(cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovariancarcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma,endometrioid tumors, Brenner tumor, clear cell carcinoma, unclassifiedcarcinoma, granulosa-theca cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma,embryonal rhabdomyosarcoma, fallopian tubes (carcinoma)); hematologiccancers (e.g., blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma)); skin cancers(e.g., malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Kaposi's sarcoma, moles, dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis); and adrenal gland cancers (e.g.,neuroblastoma).

Other aspects of the present disclosure relate to a method of increasinggenome stability of one or more human cells, by contacting the one ormore human cells with an agent that increases expression of Zscan4 inthe one or more human cells, where increased expression of Zscan4increases genome stability in the one or more human cells as compared toone or more corresponding human cells that are not contacted with theagent.

Other aspects of the present disclosure relate to a method of increasingDNA repair capacity of one or more human cells, by contacting the one ormore human cells with an agent that increases expression of Zscan4 inthe one or more human cells, where increased expression of Zscan4increases DNA repair capacity in the one or more human cells as comparedto one or more corresponding human cells that are not contacted with theagent.

Other aspects of the present disclosure relate to a method ofrejuvenating one or more human cells, by contacting the one or morehuman cells with an agent that increases expression of Zscan4 in the oneor more human cells, where increased expression of Zscan4 rejuvenatesthe one or more human cells as compared to one or more correspondinghuman cells that are not contacted with the agent.

Other aspects of the present disclosure relate to a method ofrejuvenating skin, treating atopic dermatitis, and/or a skin lesion, bytopically administering to the skin of a subject in need thereof anagent that increases expression of Zscan4.

Other aspects of the present disclosure relate to a method of treatinghair loss, by topically administering to the scalp of a subject in needthereof an agent that increases expression of Zscan4.

Other aspects of the present disclosure relate to a method of preventinghair graying, treating hair graying, or both, by administering to one ormore hair follicles of a subject in need thereof an agent that increasesexpression of Zscan4.

Other aspects of the present disclosure relate to a method ofrejuvenating a cornea, by administering to a cornea of a subject in needthereof an agent that increases expression of Zscan4.

Other aspects of the present disclosure relate to a method of treatingdry eye, by administering to a cornea of a subject in need thereof anagent that increases expression of Zscan4.

Other aspects of the present disclosure relate to a method of treatingidiopathic pulmonary fibrosis, by administering to a lung of a subjectin need thereof an agent that increases expression of Zscan4.

Other aspects of the present disclosure relate to a method of treatingatherosclerosis, a coronary heart disease, or both, by administering tothe bloodstream of a subject in need thereof an agent that increasesexpression of Zscan4.

Other aspects of the present disclosure relate to a method of providingresistance to one or more genotoxic agents in one or more human cells,by contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where increasedexpression of Zscan4 increases resistance to one or more genotoxicagents in the one or more human cells as compared to one or morecorresponding human cells that are not contacted with the agent. In someembodiments, the genotoxic agent is mitomycin C or cisplatin.

In some embodiments that may be combined with any of the precedingembodiments, the one or more human cells are human adult cells. In someembodiments, the one or more human cells are adult stem cells, tissuestem cells, progenitor cells, or induced pluripotent stem cells. In someembodiments that may be combined with any of the preceding embodiments,the one or more human cells are one or more adult stem cells, tissuestem cells, or progenitor cells selected from hematopoietic stem cells,mesenchymal stem cells, adipose stem cells, neuronal stem cells, andgerm stem cells. In some embodiments that may be combined with any ofthe preceding embodiments, the one or more human cells are somaticcells, mature cells, or differentiated cells. In some embodiments thatmay be combined with any of the preceding embodiments, the one or morehuman cells are somatic cells, mature cells, or differentiated cells. Insome embodiments that may be combined with any of the precedingembodiments, the one or more human cells are one or more somatic cells,mature cells, or differentiated cells selected from epidermal cells,fibroblasts, lymphocytes, hepatocytes, epithelial cells, myocytes,chondrocytes, osteocytes, adipocytes, cardiomyocytes, pancreatic (3cells, keratinocytes, erythrocytes, peripheral blood cells, neurocytes,astrocytes, germ cells, sperm cells, and oocytes.

Other aspects of the present disclosure relate to a method for inducinga human embryonic stem cell-like DNA methylation pattern in one or morehuman induced pluripotent stem (iPS) cells, by contacting the one ormore human iPS cells with an agent that increases expression of Zscan4in the one or more human iPS cells, where increased expression of Zscan4induces a human embryonic stem cell-like DNA methylation pattern in theone or more human iPS cells as compared to one or more correspondinghuman iPS cells that are not contacted with the agent.

Other aspects of the present disclosure relate to a method ofrejuvenating one or more human oocyte cells, by contacting the one ormore human oocyte cells with an agent that increases expression ofZscan4 in the one or more human oocyte cells, where increased expressionof Zscan4 rejuvenates the one or more human oocyte cells as compared toone or more corresponding human oocyte cells that are not contacted withthe agent.

Other aspects of the present disclosure relate to a method of increasinggenome stability of one or more human oocyte cells, by contacting theone or more human oocyte cells with an agent that increases expressionof Zscan4 in the one or more human oocyte cells, where increasedexpression of Zscan4 increases genome stability in the one or more humanoocyte cells as compared to one or more corresponding human oocyte cellsthat are not contacted with the agent. Other aspects of the presentdisclosure relate to a method of correcting one or more karyotypeabnormalities in one or more human oocyte cells, by contacting the oneor more human oocyte cells with an agent that increases expression ofZscan4 in the one or more human oocyte cells, where increased expressionof Zscan4 induces correction of the one or more karyotype abnormalitiesin the one or more human oocyte cells as compared to one or morecorresponding human oocyte cells that are not contacted with the agent.In some embodiments, the one or more human oocyte cells are isolatedfrom a subject prior to contacting with the agent that increasesexpression of Zscan4. In some embodiments, after contacting with theagent that increases expression of Zscan4 the one or more human oocytecells undergo in vitro fertilization.

Other aspects of the present disclosure relate to an in vitro method ofincreasing genome stability of one or more fertilized human oocytes, bycontacting the one or more fertilized human oocytes with an agent thatincreases expression of Zscan4 in the one or more fertilized humanoocytes, where increased expression of Zscan4 increases genome stabilityin the one or more fertilized human oocytes as compared to one or morecorresponding fertilized human oocytes embryo that are not contactedwith the agent. Other aspects of the present disclosure relate to an invitro method of correcting one or more karyotype abnormalities in one ormore fertilized human oocytes, by contacting the one or more fertilizedhuman oocytes with an agent that increases expression of Zscan4 in theone or more fertilized human oocytes, where increased expression ofZscan4 induces correction of the one or more karyotype abnormalities inthe one or more fertilized human oocytes as compared to one or morecorresponding fertilized human oocytes that are not contacted with theagent. In some embodiments, the one or more fertilized human oocyteswere fertilized by in vitro fertilization. In some embodiments, prior tobeing fertilized, the one or more human oocytes were isolated from asubject. In some embodiments, the one or more fertilized oocytes areembryos between the one-cell stage and the blastocyst stage.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a telomere abnormality, by: i.isolating human bone marrow cells from a subject suffering from adisease or condition associated with a telomere abnormality; ii.contacting the human bone marrow cells with an agent that increasesexpression of Zscan4 in the human bone marrow cells, where increasingexpression of Zscan4 induces telomere lengthening in the human bonemarrow cells; and iii. engrafting the contacted human bone marrow cellsinto the subject to treat the disease or condition associated with atelomere abnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a chromosome abnormality, by: i.isolating human bone marrow cells from a subject suffering from adisease or condition associated with a chromosome abnormality; ii.contacting the human bone marrow cells with an agent that increasesexpression of Zscan4 in the human bone marrow cells, increasingexpression of Zscan4 induces correction of the chromosome abnormality inthe human bone marrow cells; and iii. engrafting the contacted humanbone marrow cells into the subject to treat the disease or conditionassociated with a chromosome abnormality.

In some embodiments that may be combined with any of the precedingembodiments, the disease or condition is one or more diseases orconditions selected from diseases of telomere shortening, bone marrowfailure syndromes, age-related telomere shortening diseases ordisorders, and premature aging diseases or disorders. In someembodiments that may be combined with any of the preceding embodiments,the disease or condition is a disease of telomere shortening selectedfrom dyskeratosis congenita, Hoyeraal-Hreidars son syndrome, Reveszsyndrome, Coats plus syndrome, idiopathic pulmonary fibrosis, livercirrhosis, pancreatic fibrosis, Alzheimer's disease, and osteoarthritis.In some embodiments that may be combined with any of the precedingembodiments, the disease or condition is a bone marrow failure syndromeselected from Fanconi anemia, amegakaryocytic thrombocytopenia, aplasticanemia, Diamond Blackfan anemia, dyskeratosis congenita, paroxysmalnocturnal hemoglobinuria (PNH), Pearson syndrome, Shwachman Diamondsyndrome, thrombocytopenia, and myelodysplastic syndrome. In someembodiments that may be combined with any of the preceding embodiments,the disease or condition is an age-related telomere shortening diseaseor disease, a premature aging disease or disease, or both selected fromWerner syndrome, Bloom's syndrome, Hutchinson-Gilford progeria syndrome,Cockayne syndrome, Xeroderma pigmentosa, Ataxia telangiectasia, RothmundThomson syndrome, Trichothiodystrophy, Juberg-Marsidi syndrome, and Downsyndrome.

Other aspects of the present disclosure relate to a method ofrejuvenating a tissue or organ in a subject, by administering to asubject in need thereof an agent that increases expression of Zscan4 inthe tissue or organ, where increasing expression of Zscan4 rejuvenatesthe tissue or organ.

Other aspects of the present disclosure relate to a method ofrejuvenating a subject in need thereof, by administering to the subjectan agent that increases expression of Zscan4, where increasingexpression of Zscan4 rejuvenates the subject.

Other aspects of the present disclosure relate to a method of extendinglifespan of one or more human cells, by contacting the one or more humancells with an agent that increases expression of Zscan4 in one or morehuman cells in the subject, where increasing expression of Zscan4extends the lifespan of the one or more human cells as compared to oneor more corresponding human cells that are not contacted with the agent.

Other aspects of the present disclosure relate to a method of extendinglifespan of a tissue or organ in a subject, by administering to asubject in need thereof an agent that increases expression of Zscan4 inthe tissue or organ, where increasing expression of Zscan4 extends thelifespan of the tissue or organ.

Other aspects of the present disclosure relate to a method of extendinglifespan of a subject, by administering to a subject in need thereof anagent that increases expression of Zscan4 in one or more human cells inthe subject, where increasing expression of Zscan4 extends the lifespanof the one or more human cells, thereby extending the lifespan of thesubject.

Other aspects of the present disclosure relate to a method of extendinglifespan of a subject, by: i. isolating one or more human cells from thesubject; ii. contacting the one or more human cells with an agent thatincreases expression of Zscan4 in the one or more human cells, whereincreasing expression of Zscan4 extends the lifespan of the one or morehuman cells; and iii. administering the contacted one or more humancells to the subject to extend the lifespan of the subject.

Other aspects of the present disclosure relate to a method fordetermining one or more Zscan4-induced effects in one or more humancells, by: i. contacting the one or more human cells with an agent thatincreases expression of Zscan4 in one or more human cells; ii. measuringexpression levels of SERPINB4, DNMT3L, and/or DUX4 in the one or morehuman cells; and iii. comparing the expression levels of SERPINB4,DNMT3L, and/or DUX4 in the one or more human cells to the expressionlevels of SERPINB4, DNMT3L, and/or DUX4 in one or more correspondinghuman cells that are not contacted with the agent, where an increase inthe expression levels of SERPINB4, DNMT3L, and/or DUX4 in the one ormore human cells indicates the presence of one or more Zscan4-inducedeffects in the one or more human cell.

In some embodiments that may be combined with any of the precedingembodiments, the increased expression of Zscan4 is transient. In someembodiments that may be combined with any of the preceding embodiments,the agent increases Zscan4 expression for about 1 hour to about 23hours. In some embodiments that may be combined with any of thepreceding embodiments, the agent increases Zscan4 expression for about 1day to about 10 days. In some embodiments that may be combined with anyof the preceding embodiments, the agent interacts directly withendogenous Zscan4 to increase expression of Zscan4. In some embodimentsthat may be combined with any of the preceding embodiments, the agent isan isolated nucleic acid molecule encoding Zscan4. In some embodimentsthat may be combined with any of the preceding embodiments, the isolatednucleic acid molecule is a synthetic mRNA. In some embodiments that maybe combined with any of the preceding embodiments, the isolated nucleicacid molecule contains a vector. In some embodiments that may becombined with any of the preceding embodiments, the vector is a viralvector. In some embodiments that may be combined with any of thepreceding embodiments, the viral vector is a paramyxovirus vector, aretrovirus vector, a lentivirus vector or an adenovirus vector. In someembodiments that may be combined with any of the preceding embodiments,the viral vector is a paramyxovirus vector. In some embodiments that maybe combined with any of the preceding embodiments, the paramyxovirusvector is a Sendai virus vector. In some embodiments that may becombined with any of the preceding embodiments, the vector is a plasmidvector. In some embodiments that may be combined with any of thepreceding embodiments, the vector encodes Zscan4 operably linked to apromoter. In some embodiments that may be combined with any of thepreceding embodiments, the promoter is a constitutive promoter. In someembodiments that may be combined with any of the preceding embodiments,the promoter is an inducible promoter. In some embodiments that may becombined with any of the preceding embodiments, the Zscan4 is aZscan4-ERT2 fusion protein. In some embodiments that may be combinedwith any of the preceding embodiments, the Zscan4 is a Zscan4-ACprotein. In some embodiments that may be combined with any of thepreceding embodiments, the Zscan4-AC protein includes a deletion of atleast one zinc finger domain. In some embodiments that may be combinedwith any of the preceding embodiments, the Zscan4 is mouse Zscan4, humanZSCAN4, or a homolog thereof. In some embodiments that may be combinedwith any of the preceding embodiments, the Zscan4 is selected fromZscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e, and Zscan4f. In someembodiments that may be combined with any of the preceding embodiments,the isolated nucleic acid molecule contains a nucleotide sequence thatis at least 70%, at least 75%, at least 80%, at least 85%, at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to a nucleotidesequence selected from SEQ ID Nos: 1-10 and 21-30. In some embodimentsthat may be combined with any of the preceding embodiments, the Zscan4is human ZSCAN4. In some embodiments that may be combined with any ofthe preceding embodiments, the isolated nucleic acid molecule contains anucleotide sequence that is at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to SEQ ID NO: 7. In some embodiments that may be combinedwith any of the preceding embodiments, the agent is a Zscan4 protein. Insome embodiments that may be combined with any of the precedingembodiments, the Zscan4 protein is fused to a cell-penetrating peptide.In some embodiments that may be combined with any of the precedingembodiments, the cell-penetrating peptide contains a proteintransduction domain. In some embodiments that may be combined with anyof the preceding embodiments, the cell-penetrating peptide contains apoly-arginine peptide tag. In some embodiments that may be combined withany of the preceding embodiments, the Zscan4 protein is encapsulated ina nanoparticle. In some embodiments that may be combined with any of thepreceding embodiments, the Zscan4 protein is a mouse Zscan4 protein, ahuman ZSCAN4 protein, or a homolog thereof. In some embodiments that maybe combined with any of the preceding embodiments, the Zscan4 protein isselected from a Zscan4a protein, a Zscan4b protein, a Zscan4c protein, aZscan4d protein, a Zscan4e protein, and a Zscan4f protein. In someembodiments that may be combined with any of the preceding embodiments,the Zscan4 protein contains an amino acid sequence that is at least 70%,at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to an amino acid sequenceselected from SEQ ID NOs: 11-20 and 31-40. In some embodiments that maybe combined with any of the preceding embodiments, the Zscan4 protein isa human ZSCAN4 protein. In some embodiments that may be combined withany of the preceding embodiments, the Zscan4 protein contains an aminoacid sequence that is at least 70%, at least 75%, at least 80%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to SEQ ID NO: 17. In some embodiments that may be combinedwith any of the preceding embodiments, the Zscan4 protein is aZscan4-ERT2 fusion protein. In some embodiments that may be combinedwith any of the preceding embodiments, the Zscan4 protein is a Zscan4-ACprotein. In some embodiments that may be combined with any of thepreceding embodiments, the Zscan4-AC protein contains a mouse Zscan4protein, a human ZSCAN4 protein, or a homolog thereof, and where theZscan4 protein contains a deletion of at least one zinc finger domain.In some embodiments that may be combined with any of the precedingembodiments, the Zscan4-AC protein contains a Zscan4 protein selectedfrom a Zscan4a protein, a Zscan4b protein, a Zscan4c protein, a Zscan4dprotein, a Zscan4e protein, and a Zscan4f protein, and where the Zscan4protein contains a deletion of at least one zinc finger domain. In someembodiments that may be combined with any of the preceding embodiments,the Zscan4-AC protein contains a human ZSCAN4 protein, and where theZscan4 protein contains a deletion of at least one zinc finger domain.In some embodiments that may be combined with any of the precedingembodiments, the agent is a retinoid, an agent that induces oxidativestress, or both.

Other aspects of the present disclosure relate to a method of increasingtelomere length in one or more human cells, by contacting the one ormore human cells with an agent that increases expression of Zscan4 inthe human cell, where the agent is a synthetic mRNA molecule encodingZscan4 or a viral vector, preferably a Sendai viral vector, encodingZscan4, and where increased expression of Zscan4 induces telomerelengthening in the one or more human cells as compared to one or morecorresponding human cells that are not contacted with the agent.

Other aspects of the present disclosure relate to a method of treating asubject in need of telomere lengthening, by contacting one or more humancells in the subject with an agent that increases expression of Zscan4in the one or more human cells, where the agent is a synthetic mRNAmolecule encoding Zscan4 or a viral vector, preferably a Sendai viralvector, encoding Zscan4, and where increased expression of Zscan4induces telomere lengthening in the one or more human cells.

Other aspects of the present disclosure relate to a method of treating asubject in need of telomere lengthening, by: i. isolating one or morehuman cells in need of telomere lengthening from the subject; ii.contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where the agent isa synthetic mRNA molecule encoding Zscan4 or a viral vector, preferablya Sendai viral vector, encoding Zscan4, and where increasing expressionof Zscan4 induces telomere lengthening in the one or more human cells;and iii. administering the contacted one or more human cells to thesubject.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a telomere abnormality, byadministering to a subject in need thereof an agent that increasesexpression of Zscan4 in one or more human cells in the subject, wherethe agent is a synthetic mRNA molecule encoding Zscan4 or a viralvector, preferably a Sendai viral vector, encoding Zscan4, and whereincreasing expression of Zscan4 induces telomere lengthening in the oneor more human cells to treat the disease or condition associated with atelomere abnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a telomere abnormality, by: i.isolating one or more human cells from a subject suffering from adisease or condition associated with a telomere abnormality; ii.contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where the agent isa synthetic mRNA molecule encoding Zscan4 or a viral vector, preferablya Sendai viral vector, encoding Zscan4, and where increasing expressionof Zscan4 induces telomere lengthening in the one or more human cells;and iii. administering the contacted one or more human cells to thesubject to treat the disease or condition associated with a telomereabnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a chromosome abnormality, byadministering to a subject in need thereof an agent that increasesexpression of Zscan4 in one or more human cells in the subject, wherethe agent is a synthetic mRNA molecule encoding Zscan4 or a viralvector, preferably a Sendai viral vector, encoding Zscan4, and whereincreasing expression of Zscan4 induces correction of the chromosomeabnormality in the one or more human cells to treat the disease orcondition associated with a chromosome abnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a chromosome abnormality, by: i.isolating one or more human cells from a subject suffering from adisease or condition associated with a chromosome abnormality; ii.contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where the agent isa synthetic mRNA molecule encoding Zscan4 or a viral vector, preferablya Sendai viral vector, encoding Zscan4, and where increasing expressionof Zscan4 induces correction of the chromosome abnormality in the one ormore human cells; and iii. administering the contacted one or more humancells to the subject to treat the disease or condition associated with achromosome abnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a karyotype abnormality, byadministering to a subject in need thereof an agent that increasesexpression of Zscan4 in one or more human cells in the subject, wherethe agent is a synthetic mRNA molecule encoding Zscan4 or a viralvector, preferably a Sendai viral vector, encoding Zscan4, and whereincreasing expression of Zscan4 induces correction of the karyotypeabnormality in the one or more human cells to treat the disease orcondition associated with a karyotype abnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a karyotype abnormality, by: i.isolating one or more human cells from a subject suffering from adisease or condition associated with a karyotype abnormality; ii.contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where the agent isa synthetic mRNA molecule encoding Zscan4 or a viral vector, preferablya Sendai viral vector, encoding Zscan4, and where increasing expressionof Zscan4 induces correction of the karyotype abnormality in the one ormore human cells; and iii. administering the contacted one or more humancells to the subject to treat the disease or condition associated with akaryotype abnormality.

In some embodiments that may be combined with any of the precedingembodiments, the karyotype abnormality is selected from a chromosomenullisomy, a chromosome monosomy, a chromosome trisomy, a chromosometetrasomy, and a chromosome pentasomy. In some embodiments that may becombined with any of the preceding embodiments, the karyotypeabnormality is selected from trisomy 21, trisomy 16, trisomy 18, trisomy13, monosomy X, XXX aneuploidy, XXY aneuploidy, XYY aneuploidy, and 1p36duplication. In some embodiments that may be combined with any of thepreceding embodiments, the disease or condition associated with akaryotype abnormality is selected from dup(17)(p11.2p11.2) syndrome,Pelizaeus-Merzbacher disease, dup(22)(q11.2q11.2) syndrome, cat-eyesyndrome, Cri-du-chat syndrome, Wolf-Hirschhorn, Williams-Beurensyndrome, Charcot-Marie-Tooth disease, Hereditary neuropathy withliability to pressure palsies, Smith-Magenis syndrome,Neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome,DiGeorge syndrome, Steroid sulfatase deficiency, Kallmann syndrome,Microphthalmia with linear skin defects, Adrenal hypoplasia, Glycerolkinase deficiency, Pelizaeus-Merzbacher disease, Testis-determiningfactor on Y, Azoospermia (factor a), Azoospermia (factor b), Azoospermia(factor c), and 1p36 deletion. In some embodiments that may be combinedwith any of the preceding embodiments, the disease or condition is oneor more diseases or conditions selected from diseases of telomereshortening, bone marrow failure syndromes, age-related telomereshortening diseases or disorders, and premature aging diseases ordisorders. In some embodiments that may be combined with any of thepreceding embodiments, the disease or condition is a disease of telomereshortening selected from dyskeratosis congenita, Hoyeraal-Hreidars sonsyndrome, Revesz syndrome, Coats plus syndrome, idiopathic pulmonaryfibrosis, liver cirrhosis, pancreatic fibrosis, Alzheimer's disease, andosteoarthritis. In some embodiments that may be combined with any of thepreceding embodiments, the disease or condition is a bone marrow failuresyndrome selected from Fanconi anemia, amegakaryocytic thrombocytopenia,aplastic anemia, Diamond Blackfan anemia, dyskeratosis congenita,paroxysmal nocturnal hemoglobinuria (PNH), Pearson syndrome, ShwachmanDiamond syndrome, thrombocytopenia, and myelodysplastic syndrome. Insome embodiments that may be combined with any of the precedingembodiments, the disease or condition is an age-related telomereshortening disease or disorder, a premature aging disease or disorder,or both selected from Werner syndrome, Bloom's syndrome,Hutchinson-Gilford progeria syndrome, Cockayne syndrome, Xerodermapigmentosa, Ataxia telangiectasia, Rothmund Thomson syndrome,Trichothiodystrophy, Juberg-Marsidi syndrome, and Down syndrome. In someembodiments that may be combined with any of the preceding embodiments,the disease or condition is one or more diseases or conditions selectedfrom immunological deficiencies, an autoimmune disease, an autoimmunedisorder, chronic ulcers, atherosclerosis, cancer, a neurologic injury,a degenerative disorder, a neurodegenerative disorder, wound healing,muscle repair, cardiac muscle repair, cartilage replacement, arthritis,osteoarthritis, tooth regeneration, blindness, age-related blindness dueto proliferative decline of retinal pigmented epithelial cells,deafness, bone marrow failure, bone marrow transplant, diabetes,muscular dystrophy, Duchenne muscular dystrophy, a genetic disease, agenetic mutation, and DNA damage. In some embodiments that may becombined with any of the preceding embodiments, the disease or conditionis a cancer selected from cancers of the heart (e.g., angiosarcoma,fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma,fibroma, lipoma and teratoma), lung cancers (e.g., bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma);gastrointestinal tract cancers (e.g., esophagus (squamous cellcarcinoma, adenocarcinoma, leiomyosarcoma, lymphoma); stomach cancers(carcinoma, lymphoma, leiomyosarcoma); pancreatic cancers (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma); small bowel cancers (adenocarcinoma, lymphoma, carcinoidtumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma); large bowel cancers (adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma); genitourinary tract cancers (e.g.,kidney (adenocarcinoma, Wilms' tumor, nephroblastoma, lymphoma,leukemia); bladder and urethra cancers (squamous cell carcinoma,transitional cell carcinoma, adenocarcinoma); prostate cancers(adenocarcinoma, sarcoma); testis cancers (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); livercancers (e.g., hepatoma (hepatocellular carcinoma), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma); bonecancers (e.g., osteogenic sarcoma (osteosarcoma), fibrosarcoma,malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma,malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignantgiant cell tumor, chordoma, osteochondroma (osteocartilaginousexostoses), benign chondroma, chondroblastoma, chondromyxofibroma,osteoid osteoma and giant cell tumors); nervous system cancers (e.g.,skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans),meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma, pinealoma, glioblastomamultiforme, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma));gynecological cancers (e.g., uterus (endometrial carcinoma), cervix(cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovariancarcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma,endometrioid tumors, Brenner tumor, clear cell carcinoma, unclassifiedcarcinoma, granulosa-theca cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma,embryonal rhabdomyosarcoma, fallopian tubes (carcinoma)); hematologiccancers (e.g., blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma)); skin cancers(e.g., malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Kaposi's sarcoma, moles, dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis); and adrenal gland cancers (e.g.,neuroblastoma). In some embodiments that may be combined with any of thepreceding embodiments, the disease or condition is an autoimmune diseaseselected from thyroiditis, Goodpasture's disease, rheumatoid arthritis,juvenile oligoarthritis, collagen-induced arthritis, adjuvant-inducedarthritis, Sjogren's syndrome, multiple sclerosis, experimentalautoimmune encephalomyelitis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, autoimmune gastric atrophy, pemphigusvulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes,myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosingcholangitis, sclerosing sialadenitis, systemic lupus erythematosis,autoimmune thrombocytopenia purpura, Addison's disease, systemicsclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia,and pernicious anemia. In some embodiments that may be combined with anyof the preceding embodiments, the disease or condition is aneurodegenerative disease selected from adrenoleukodystrophy (ALD),alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease,amyotrophic lateral sclerosis, Lou Gehrig's Disease, ataxiatelangiectasia, Batten disease, Spielmeyer-Vogt-Sjogren-Batten disease,bovine spongiform encephalopathy (BSE), Canavan disease, cerebral palsy,Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,familial fatal insomnia, frontotemporal lobar degeneration, Huntington'sdisease, HIV-associated dementia, Kennedy's disease, Krabbe's disease,Lewy body dementia, neuroborreliosis, Machado-Joseph disease,Spinocerebellar ataxia type 3, Multiple System Atrophy, multiplesclerosis, narcolepsy, Niemann Pick disease, Parkinson's disease,Pelizaeus-Merzbacher Disease, Pick's disease, primary lateral sclerosis,prion diseases, progressive supranuclear palsy, Refsum's disease,Sandhoff disease, Schilder's disease, subacute combined degeneration ofspinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease, Batten disease, spinocerebellarataxia, spinal muscular atrophy, Steele-Richardson-Olszewski disease,Tabes dorsalis, and toxic encephalopathy.

Other aspects of the present disclosure relate to a method of treating acancer, by administering to a subject in need thereof an agent thatincreases expression of Zscan4 in one or more cancer cells in thesubject, where the agent is a synthetic mRNA molecule encoding Zscan4 ora viral vector, preferably a Sendai viral vector, encoding Zscan4, andwhere increasing expression of Zscan4 represses growth of the one ormore cancer cells, thereby treating the cancer. Other aspects of thepresent disclosure relate to a method of improving responsiveness tochemotherapy in a cancer patient, by administering to a subject in needthereof an agent that reduces expression of endogenous ZSCAN4 in one ormore cancer stem cells in the subject, where the agent is a syntheticmRNA molecule encoding Zscan4 or a viral vector, preferably a Sendaiviral vector, encoding Zscan4, and where reducing expression ofendogenous ZSCAN4 reduces or eliminates resistance to one or morechemotherapeutic agents in the one or more cancer stem cells, therebyimproving responsiveness to the one or more chemotherapeutic agents inthe subject. In some embodiments, the agent that reduces expression ofendogenous ZSCAN4 is an siRNA or shRNA specific for ZSCAN4. In someembodiments, the cancer selected from cancers of the heart (e.g.angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma,rhabdomyoma, fibroma, lipoma and teratoma), lung cancers (e.g.,bronchogenic carcinoma (squamous cell, undifferentiated small cell,undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma); gastrointestinal tract cancers (e.g.,esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma,lymphoma); stomach cancers (carcinoma, lymphoma, leiomyosarcoma);pancreatic cancers (ductal adenocarcinoma, insulinoma, glucagonoma,gastrinoma, carcinoid tumors, vipoma); small bowel cancers(adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma); large bowelcancers (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); genitourinary tract cancers (e.g., kidney (adenocarcinoma,Wilms' tumor, nephroblastoma, lymphoma, leukemia); bladder and urethracancers (squamous cell carcinoma, transitional cell carcinoma,adenocarcinoma); prostate cancers (adenocarcinoma, sarcoma); testiscancers (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma); liver cancers (e.g., hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma); bone cancers (e.g.,osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor,chordoma, osteochondroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors); nervous system cancers (e.g., skull (osteoma,hemangioma, granuloma, xanthoma, osteitis deformans), meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma, pinealoma, glioblastomamultiforme, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma));gynecological cancers (e.g., uterus (endometrial carcinoma), cervix(cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovariancarcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma,endometrioid tumors, Brenner tumor, clear cell carcinoma, unclassifiedcarcinoma, granulosa-theca cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma,embryonal rhabdomyosarcoma, fallopian tubes (carcinoma)); hematologiccancers (e.g., blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma)); skin cancers(e.g., malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Kaposi's sarcoma, moles, dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis); and adrenal gland cancers (e.g.,neuroblastoma).

Other aspects of the present disclosure relate to a method of increasinggenome stability of one or more human cells, by contacting the one ormore human cells with an agent that increases expression of Zscan4 inthe one or more human cells, where the agent is a synthetic mRNAmolecule encoding Zscan4 or a viral vector, preferably a Sendai viralvector, encoding Zscan4, and where increased expression of Zscan4increases genome stability in the one or more human cells as compared toone or more corresponding human cells that are not contacted with theagent.

Other aspects of the present disclosure relate to a method of increasingDNA repair capacity of one or more human cells, by contacting the one ormore human cells with an agent that increases expression of Zscan4 inthe one or more human cells, where the agent is a synthetic mRNAmolecule encoding Zscan4 or a viral vector, preferably a Sendai viralvector, encoding Zscan4, and where increased expression of Zscan4increases DNA repair capacity in the one or more human cells as comparedto one or more corresponding human cells that are not contacted with theagent.

Other aspects of the present disclosure relate to a method ofrejuvenating one or more human cells, by contacting the one or morehuman cells with an agent that increases expression of Zscan4 in the oneor more human cells, where the agent is a synthetic mRNA moleculeencoding Zscan4 or a viral vector, preferably a Sendai viral vector,encoding Zscan4, and where increased expression of Zscan4 rejuvenatesthe one or more human cells as compared to one or more correspondinghuman cells that are not contacted with the agent.

Other aspects of the present disclosure relate to a method ofrejuvenating skin, treating atopic dermatitis, and/or a skin lesion, bytopically administering to the skin of a subject in need thereof anagent that increases expression of Zscan4, where the agent is asynthetic mRNA molecule encoding Zscan4 or a viral vector, preferably aSendai viral vector, encoding Zscan4.

Other aspects of the present disclosure relate to a method of treatinghair loss, by topically administering to the scalp of a subject in needthereof an agent that increases expression of Zscan4, where the agent isa synthetic mRNA molecule encoding Zscan4 or a viral vector, preferablya Sendai viral vector, encoding Zscan4.

Other aspects of the present disclosure relate to a method of preventinghair graying, treating hair graying, or both, by administering to one ormore hair follicles of a subject in need thereof an agent that increasesexpression of Zscan4, where the agent is a synthetic mRNA moleculeencoding Zscan4 or a viral vector, preferably a Sendai viral vector,encoding Zscan4.

Other aspects of the present disclosure relate to a method ofrejuvenating a cornea, by administering to a cornea of a subject in needthereof an agent that increases expression of Zscan4, where the agent isa synthetic mRNA molecule encoding Zscan4 or a viral vector, preferablya Sendai viral vector, encoding Zscan4.

Other aspects of the present disclosure relate to a method of treatingdry eye, by administering to a cornea of a subject in need thereof anagent that increases expression of Zscan4, where the agent is asynthetic mRNA molecule encoding Zscan4 or a viral vector, preferably aSendai viral vector, encoding Zscan4.

Other aspects of the present disclosure relate to a method of treatingidiopathic pulmonary fibrosis, by administering to a lung of a subjectin need thereof an agent that increases expression of Zscan4, where theagent is a synthetic mRNA molecule encoding Zscan4 or a viral vector,preferably a Sendai viral vector, encoding Zscan4.

Other aspects of the present disclosure relate to a method of treatingatherosclerosis, a coronary heart disease, or both, by administering tothe bloodstream of a subject in need thereof an agent that increasesexpression of Zscan4, where the agent is a synthetic mRNA moleculeencoding Zscan4 or a viral vector, preferably a Sendai viral vector,encoding Zscan4.

Other aspects of the present disclosure relate to a method of providingresistance to one or more genotoxic agents in one or more human cells,by contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where the agent isa synthetic mRNA molecule encoding Zscan4 or a viral vector, preferablya Sendai viral vector, encoding Zscan4, and where increased expressionof Zscan4 increases resistance to one or more genotoxic agents in theone or more human cells as compared to one or more corresponding humancells that are not contacted with the agent. In some embodiments, thegenotoxic agent is mitomycin C or cisplatin.

In some embodiments that may be combined with any of the precedingembodiments, the one or more human cells are human adult cells. In someembodiments that may be combined with any of the preceding embodiments,the one or more human cells are adult stem cells, tissue stem cells,progenitor cells, or induced pluripotent stem cells. In some embodimentsthat may be combined with any of the preceding embodiments, the one ormore human cells are one or more adult stem cells, tissue stem cells, orprogenitor cells selected from hematopoietic stem cells, mesenchymalstem cells, adipose stem cells, neuronal stem cells, and germ stemcells. In some embodiments that may be combined with any of thepreceding embodiments, the one or more human cells are somatic cells,mature cells, or differentiated cells. In some embodiments that may becombined with any of the preceding embodiments, the one or more humancells are somatic cells, mature cells, or differentiated cells. In someembodiments that may be combined with any of the preceding embodiments,the one or more human cells are one or more somatic cells, mature cells,or differentiated cells selected from epidermal cells, fibroblasts,lymphocytes, hepatocytes, epithelial cells, myocytes, chondrocytes,osteocytes, adipocytes, cardiomyocytes, pancreatic β cells,keratinocytes, erythrocytes, peripheral blood cells, neurocytes,astrocytes, germ cells, sperm cells, and oocytes.

Other aspects of the present disclosure relate to a method for inducinga human embryonic stem cell-like DNA methylation pattern in one or morehuman induced pluripotent stem (iPS) cells, by contacting the one ormore human iPS cells with an agent that increases expression of Zscan4in the one or more human iPS cells, where the agent is a synthetic mRNAmolecule encoding Zscan4 or a viral vector, preferably a Sendai viralvector, encoding Zscan4, and where increased expression of Zscan4induces a human embryonic stem cell-like DNA methylation pattern in theone or more human iPS cells as compared to one or more correspondinghuman iPS cells that are not contacted with the agent.

Other aspects of the present disclosure relate to a method ofrejuvenating one or more human oocyte cells, by contacting the one ormore human oocyte cells with an agent that increases expression ofZscan4 in the one or more human oocyte cells, where the agent is asynthetic mRNA molecule encoding Zscan4 or a viral vector, preferably aSendai viral vector, encoding Zscan4, and where increased expression ofZscan4 rejuvenates the one or more human oocyte cells as compared to oneor more corresponding human oocyte cells that are not contacted with theagent.

Other aspects of the present disclosure relate to a method of increasinggenome stability of one or more human oocyte cells, by contacting theone or more human oocyte cells with an agent that increases expressionof Zscan4 in the one or more human oocyte cells, where the agent is asynthetic mRNA molecule encoding Zscan4 or a viral vector, preferably aSendai viral vector, encoding Zscan4, and where increased expression ofZscan4 increases genome stability in the one or more human oocyte cellsas compared to one or more corresponding human oocyte cells that are notcontacted with the agent. Other aspects of the present disclosure relateto a method of correcting one or more karyotype abnormalities in one ormore human oocyte cells, by contacting the one or more human oocytecells with an agent that increases expression of Zscan4 in the one ormore human oocyte cells, where the agent is a synthetic mRNA moleculeencoding Zscan4 or a viral vector, preferably a Sendai viral vector,encoding Zscan4, and where increased expression of Zscan4 inducescorrection of the one or more karyotype abnormalities in the one or morehuman oocyte cells as compared to one or more corresponding human oocytecells that are not contacted with the agent. In some embodiments, theone or more human oocyte cells are isolated from a subject prior tocontacting with the agent that increases expression of Zscan4. In someembodiments, after contacting with the agent that increases expressionof Zscan4 the one or more human oocyte cells undergo in vitrofertilization.

Other aspects of the present disclosure relate to an in vitro method ofincreasing genome stability of one or more fertilized human oocytes, bycontacting the one or more fertilized human oocytes with an agent thatincreases expression of Zscan4 in the one or more fertilized humanoocytes, where the agent is a synthetic mRNA molecule encoding Zscan4 ora viral vector, preferably a Sendai viral vector, encoding Zscan4, andwhere increased expression of Zscan4 increases genome stability in theone or more fertilized human oocytes as compared to one or morecorresponding fertilized human oocytes embryo that are not contactedwith the agent. Other aspects of the present disclosure relate to an invitro method of correcting one or more karyotype abnormalities in one ormore fertilized human oocytes, by contacting the one or more fertilizedhuman oocytes with an agent that increases expression of Zscan4 in theone or more fertilized human oocytes, where the agent is a syntheticmRNA molecule encoding Zscan4 or a viral vector, preferably a Sendaiviral vector, encoding Zscan4, and where increased expression of Zscan4induces correction of the one or more karyotype abnormalities in the oneor more fertilized human oocytes as compared to one or morecorresponding fertilized human oocytes that are not contacted with theagent. In some embodiments, the one or more fertilized human oocyteswere fertilized by in vitro fertilization. In some embodiments, prior tobeing fertilized, the one or more human oocytes were isolated from asubject. In some embodiments, the one or more fertilized oocytes areembryos between the one-cell stage and the blastocyst stage.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a telomere abnormality, by: i.isolating human bone marrow cells from a subject suffering from adisease or condition associated with a telomere abnormality; ii.contacting the human bone marrow cells with an agent that increasesexpression of Zscan4 in the human bone marrow cells, where the agent isa synthetic mRNA molecule encoding Zscan4 or a viral vector, preferablya Sendai viral vector, encoding Zscan4, and where increasing expressionof Zscan4 induces telomere lengthening in the human bone marrow cells;and iii. engrafting the contacted human bone marrow cells into thesubject to treat the disease or condition associated with a telomereabnormality.

Other aspects of the present disclosure relate to a method of treating adisease or condition associated with a chromosome abnormality, by: i.isolating human bone marrow cells from a subject suffering from adisease or condition associated with a chromosome abnormality; ii.contacting the human bone marrow cells with an agent that increasesexpression of Zscan4 in the human bone marrow cells, where the agent isa synthetic mRNA molecule encoding Zscan4 or a viral vector, preferablya Sendai viral vector, encoding Zscan4, and where increasing expressionof Zscan4 induces correction of the chromosome abnormality in the humanbone marrow cells; and iii. engrafting the contacted human bone marrowcells into the subject to treat the disease or condition associated witha chromosome abnormality.

In some embodiments that may be combined with any of the precedingembodiments, the disease or condition is one or more diseases orconditions selected from diseases of telomere shortening, bone marrowfailure syndromes, age-related telomere shortening diseases ordisorders, and premature aging diseases or disorders. In someembodiments that may be combined with any of the preceding embodiments,the disease or condition is a disease of telomere shortening selectedfrom dyskeratosis congenita, Hoyeraal-Hreidars son syndrome, Reveszsyndrome, Coats plus syndrome, idiopathic pulmonary fibrosis, livercirrhosis, pancreatic fibrosis, Alzheimer's disease, and osteoarthritis.In some embodiments that may be combined with any of the precedingembodiments, the disease or condition is a bone marrow failure syndromeselected from Fanconi anemia, amegakaryocytic thrombocytopenia, aplasticanemia, Diamond Blackfan anemia, dyskeratosis congenita, paroxysmalnocturnal hemoglobinuria (PNH), Pearson syndrome, Shwachman Diamondsyndrome, thrombocytopenia, and myelodysplastic syndrome. In someembodiments that may be combined with any of the preceding embodiments,the disease or condition is an age-related telomere shortening diseaseor disease, a premature aging disease or disease, or both selected fromWerner syndrome, Bloom's syndrome, Hutchinson-Gilford progeria syndrome,Cockayne syndrome, Xeroderma pigmentosa, Ataxia telangiectasia, RothmundThomson syndrome, Trichothiodystrophy, Juberg-Marsidi syndrome, and Downsyndrome.

Other aspects of the present disclosure relate to a method ofrejuvenating a tissue or organ in a subject, by administering to asubject in need thereof an agent that increases expression of Zscan4 inthe tissue or organ, where the agent is a synthetic mRNA moleculeencoding Zscan4 or a viral vector, preferably a Sendai viral vector,encoding Zscan4, and where increasing expression of Zscan4 rejuvenatesthe tissue or organ.

Other aspects of the present disclosure relate to a method ofrejuvenating a subject in need thereof, by administering to the subjectan agent that increases expression of Zscan4, where the agent is asynthetic mRNA molecule encoding Zscan4 or a viral vector, preferably aSendai viral vector, encoding Zscan4, and where increasing expression ofZscan4 rejuvenates the subject.

Other aspects of the present disclosure relate to a method of extendinglifespan of one or more human cells, by contacting the one or more humancells with an agent that increases expression of Zscan4 in one or morehuman cells in the subject, where the agent is a synthetic mRNA moleculeencoding Zscan4 or a viral vector, preferably a Sendai viral vector,encoding Zscan4, and where increasing expression of Zscan4 extends thelifespan of the one or more human cells as compared to one or morecorresponding human cells that are not contacted with the agent.

Other aspects of the present disclosure relate to a method of extendinglifespan of a tissue or organ in a subject, by administering to asubject in need thereof an agent that increases expression of Zscan4 inthe tissue or organ, where the agent is a synthetic mRNA moleculeencoding Zscan4 or a viral vector, preferably a Sendai viral vector,encoding Zscan4, and where increasing expression of Zscan4 extends thelifespan of the tissue or organ.

Other aspects of the present disclosure relate to a method of extendinglifespan of a subject, by administering to a subject in need thereof anagent that increases expression of Zscan4 in one or more human cells inthe subject, where the agent is a synthetic mRNA molecule encodingZscan4 or a viral vector, preferably a Sendai viral vector, encodingZscan4, and where increasing expression of Zscan4 extends the lifespanof the one or more human cells, thereby extending the lifespan of thesubject.

Other aspects of the present disclosure relate to a method of extendinglifespan of a subject, by: i. isolating one or more human cells from thesubject; ii. contacting the one or more human cells with an agent thatincreases expression of Zscan4 in the one or more human cells, where theagent is a synthetic mRNA molecule encoding Zscan4 or a viral vector,preferably a Sendai viral vector, encoding Zscan4, and where increasingexpression of Zscan4 extends the lifespan of the one or more humancells; and iii. administering the contacted one or more human cells tothe subject to extend the lifespan of the subject.

Other aspects of the present disclosure relate to a method fordetermining one or more Zscan4-induced effects in one or more humancells, by: i. contacting the one or more human cells with an agent thatincreases expression of Zscan4 in one or more human cells, where theagent is a synthetic mRNA molecule encoding Zscan4 or a viral vector,preferably a Sendai viral vector, encoding Zscan4; ii. measuringexpression levels of SERPINB4, DNMT3L, and/or DUX4 in the one or morehuman cells; and iii. comparing the expression levels of SERPINB4,DNMT3L, and/or DUX4 in the one or more human cells to the expressionlevels of SERPINB4, DNMT3L, and/or DUX4 in one or more correspondinghuman cells that are not contacted with the agent, where an increase inthe expression levels of SERPINB4, DNMT3L, and/or DUX4 in the one ormore human cells indicates the presence of one or more Zscan4-inducedeffects in the one or more human cell.

In some embodiments that may be combined with any of the precedingembodiments, the increased expression of Zscan4 is transient. In someembodiments that may be combined with any of the preceding embodiments,the agent increases Zscan4 expression for about 1 hour to about 23hours. In some embodiments that may be combined with any of thepreceding embodiments, the agent increases Zscan4 expression for about 1day to about 10 days. In some embodiments that may be combined with anyof the preceding embodiments, the agent interacts directly withendogenous Zscan4 to increase expression of Zscan4. In some embodimentsthat may be combined with any of the preceding embodiments, the vectorencodes Zscan4 operably linked to a promoter. In some embodiments thatmay be combined with any of the preceding embodiments, the promoter is aconstitutive promoter. In some embodiments that may be combined with anyof the preceding embodiments, the promoter is an inducible promoter. Insome embodiments that may be combined with any of the precedingembodiments, the Zscan4 is a Zscan4-ERT2 fusion protein. In someembodiments that may be combined with any of the preceding embodiments,the Zscan4 is a Zscan4-AC protein. In some embodiments that may becombined with any of the preceding embodiments, the Zscan4-AC proteinincludes a deletion of at least one zinc finger domain. In someembodiments that may be combined with any of the preceding embodiments,the Zscan4 is mouse Zscan4, human ZSCAN4, or a homolog thereof. In someembodiments that may be combined with any of the preceding embodiments,the Zscan4 is selected from Zscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e,and Zscan4f. In some embodiments that may be combined with any of thepreceding embodiments, the isolated nucleic acid molecule contains anucleotide sequence that is at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to a nucleotide sequence selected from SEQ ID Nos: 1-10and 21-30. In some embodiments that may be combined with any of thepreceding embodiments, the Zscan4 is human ZSCAN4. In some embodimentsthat may be combined with any of the preceding embodiments, the isolatednucleic acid molecule contains a nucleotide sequence that is at least70%, at least 75%, at least 80%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7. Insome embodiments that may be combined with any of the precedingembodiments, the agent is a Zscan4 protein. In some embodiments that maybe combined with any of the preceding embodiments, the Zscan4 protein isfused to a cell-penetrating peptide. In some embodiments that may becombined with any of the preceding embodiments, the cell-penetratingpeptide contains a protein transduction domain. In some embodiments thatmay be combined with any of the preceding embodiments, thecell-penetrating peptide contains a poly-arginine peptide tag. In someembodiments that may be combined with any of the preceding embodiments,the Zscan4 protein is encapsulated in a nanoparticle. In someembodiments that may be combined with any of the preceding embodiments,the Zscan4 protein is a mouse Zscan4 protein, a human ZSCAN4 protein, ora homolog thereof. In some embodiments that may be combined with any ofthe preceding embodiments, the Zscan4 protein is selected from a Zscan4aprotein, a Zscan4b protein, a Zscan4c protein, a Zscan4d protein, aZscan4e protein, and a Zscan4f protein. In some embodiments that may becombined with any of the preceding embodiments, the Zscan4 proteincontains an amino acid sequence that is at least 70%, at least 75%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to an amino acid sequence selected from SEQID NOs: 11-20 and 31-40. In some embodiments that may be combined withany of the preceding embodiments, the Zscan4 protein is a human ZSCAN4protein. In some embodiments that may be combined with any of thepreceding embodiments, the Zscan4 protein contains an amino acidsequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto SEQ ID NO: 17. In some embodiments that may be combined with any ofthe preceding embodiments, the Zscan4 protein is a Zscan4-ERT2 fusionprotein. In some embodiments that may be combined with any of thepreceding embodiments, the Zscan4 protein is a Zscan4-AC protein. Insome embodiments that may be combined with any of the precedingembodiments, the Zscan4-AC protein contains a mouse Zscan4 protein, ahuman ZSCAN4 protein, or a homolog thereof, and where the Zscan4 proteincontains a deletion of at least one zinc finger domain. In someembodiments that may be combined with any of the preceding embodiments,the Zscan4-AC protein contains a Zscan4 protein selected from a Zscan4aprotein, a Zscan4b protein, a Zscan4c protein, a Zscan4d protein, aZscan4e protein, and a Zscan4f protein, and where the Zscan4 proteincontains a deletion of at least one zinc finger domain. In someembodiments that may be combined with any of the preceding embodiments,the Zscan4-AC protein contains a human ZSCAN4 protein, and where theZscan4 protein contains a deletion of at least one zinc finger domain.In some embodiments that may be combined with any of the precedingembodiments, the agent is a retinoid, an agent that induces oxidativestress, or both.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B depict the correction of chromosome abnormalities in mouse EScells transfected with hZSCAN4-mRNAs. FIG. 1A depicts the experimentalprocedure. FIG. 1B depicts the percent of euploid mouse ES cellstransfected with hZSCAN4 mRNAs.

FIGS. 2A-B depict the correction of chromosome abnormalities in mouse EScells infected with Sendai virus vectors expressing mZscan4 or hZSCAN4.FIG. 2A depicts the percent of euploid mouse ES cells infected withSeVmZscan4 or SeVhZSCAN4. FIG. 2B depicts the percent of euploid mouseES cells infected with SeVmZERT2 or SeVhZERT2.

FIGS. 3A-C depict the correction of chromosome abnormalities in mouse EScells infected with temperature-sensitive Sendai virus vectorsexpressing mZscan4 or hZSCAN4. FIG. 3A depicts the percent of euploidmouse ES cells infected with SeVmZscan4-TS15 or SeVhZSCAN4-TS15,followed by culturing at 35° C. for three days. FIG. 3B depicts thepercent of euploid mouse ES cells infected with SeVmZscan4-TS15 orSeVhZSCAN4-TS15 followed by culturing at 35° C. for six days. FIG. 3Cdepicts the percent of euploid mouse ES cells infected withSeVmZscan4-TS15 or SeVhZSCAN4-TS15, followed by culturing at 35° C. forthree days and then at 37° C. for three days.

FIGS. 4A-C depict the effects of Zscan4 biologics on mouse ES cells.FIG. 4A depicts the effects of transfecting mouse ES cells with hZSCAN4mRNAs. FIG. 4B depicts the effects of infecting mouse ES cells withSendai virus vectors expressing Zscan4. FIG. 4C depicts the effects ofinfecting mouse ES cells with temperature-sensitive Sendai virus vectorsexpressing mZscan4 or hZSCAN4.

FIGS. 5A-C depict the effects of Zscan4 biologics on human iPS cells.FIG. 5A depicts the effects of transfecting human iPS cells with Zscan4mRNAs. FIG. 5B depicts the effects of infecting human iPS cells withSendai virus vectors expressing Zscan4. FIG. 5C depicts the effects ofinfecting human iPS cells with temperature-sensitive Sendai virusvectors expressing mZscan4 or hZSCAN4.

FIG. 6 depicts the results of a growth assay of human fibroblast cellsfrom a DKC patient transfected with hZSCAN4 mRNAs or GFP mRNAs.

FIG. 7A depicts the results of a growth assay of human fibroblast cellsfrom a DKC patient infected with SeVhZScan4. FIG. 7B shows micrographsdepicting the cell morphology of human fibroblast cells from a DKCpatient infected with SeVhZScan4.

FIG. 8A depicts the experimental procedure. FIG. 8B depicts the resultsof a telomere length assay of human fibroblast cells from a DKC patienttransfected with hZSCAN4 mRNAs or GFP mRNAs.

FIG. 9 depicts the results of a growth assay of fibroblast cells of aWerner syndrome (WS) patient transfected with hZSCAN4 mRNAs or GFPmRNAs.

FIG. 10 depicts an exemplary treatment scheme using Zscan4.

FIG. 11 depicts a bar graph showing that overexpression of human ZSCAN4increases telomere length in normal adult human fibroblast cells. “N”indicates the number of replicates.

FIG. 12 depicts a bar graph showing that overexpression of human ZSCAN4increases telomere length in human fibroblasts isolated from a patientwith Fanconi anemia, complementation group A. “N” indicates the numberof replicates.

FIGS. 13A-C depict the results of growth assays of human adult dermalfibroblast (HDFa) cells transfected with hZSCAN4 mRNAs, mZscan4 mRNAs,or GFP mRNAs. FIG. 13A depicts the results of a growth assay of HDFacells transfected with hZSCAN4 mRNAs or GFP mRNAs and cultured forapproximately 50 days. FIG. 13B depicts the results of a growth assay ofHDFa cells transfected with hZSCAN4 mRNAs or GFP mRNAs and cultured forapproximately 30 days. FIG. 13C depicts the results of a growth assay ofHDFa cells transfected with mZscan4 mRNAs, hZSCAN4 mRNAs, or GFP mRNAsand cultured for approximately 20 days.

FIG. 14 depicts the results of extension of telomere lengths in humanmesenchymal stem (MS) cells infected with temperature-sensitive Sendaivirus vectors expressing mouse Zscan4 or human ZSCAN4.

FIG. 15 depicts the effects of Zscan4 biologics on differentiated cellsand tissue stem cells.

FIGS. 16A-C depict the ploidy number of chromosome 21 from fibroblastcells from a Down syndrome patient (DS cells) transfected with hZSCAN4mRNAs. FIG. 16A depicts typical results of FISH analyses. Three dotsindicate trisomy 21 and two dots indicate normal diploid chromosome 21.FIG. 16B depicts DS cells transfected once with hZSCAN4 mRNAs.

FIG. 16C depicts DS cells transfected twice with hZSCAN4 mRNAs. In thefigure, “n” indicates the number of examined nuclei.

FIG. 17A depicts the experimental procedure. FIG. 17B depicts the ploidynumber of chromosome 21 from fibroblast cells from a Down Syndromepatient (DS cells) infected with SeVhZSCAN4-TS15 once, followed byinfection with SeVhZSCAN4 twice. FIG. 17C depicts the ploidy number ofchromosome 21 from fibroblast cells from a Down Syndrome patient (DScells) infected with SeVhZSCAN4-TS15 once, followed by infection withSeVhZSCAN4 four times.

FIG. 18 depicts an exemplary treatment scheme for rejuvenating and/orcorrecting chromosome abnormalities in human oocytes, human fertilizedoocytes, and human preimplantation embryos using mouse Zscan4 or humanZSCAN4.

FIG. 19 depicts the repression of cell growth of HCT116 cancer cellsinfected with either SeVmZscan4-TS15 or SeVhZSCAN4-TS15.

DETAILED DESCRIPTION Overview

As discussed above, it has been previously shown that expression ofmouse Zscan4 in mouse embryonic stem cells is associated telomereelongation. Given that the mouse genome contains six Zscan4 genes andthree Zscan4 pseudogenes, while the human genome only contains oneZscan4 gene, one of ordinary skill in the art would not have been ableto extrapolate the results of Zscan4 expression in mouse cells to humancells. However, as disclosed in the Example 8 below, applicant has forthe first time shown that expression of human ZSCAN4 in fullydifferentiated, adult human fibroblasts results in about 40% increase intelomere length in the fibroblasts. Moreover, applicant has shown thatexpression of Zscan4 in human fibroblasts isolated from a patient withFanconi anemia resulted in about a 160% increase in telomere length inthe fibroblasts. These results surprisingly demonstrate that Zscan4 isan upstream effector, rather than a downstream actor in telomereelongation, as Zscan4 expression alone was shown to be sufficient toincrease telomere length in human fibroblasts isolated from a patientwith Fanconi anemia. As such, activating or increasing expression ofZscan4 in cells can be an effective treatment for Fanconi anemia or anyother disease or condition associated with telomere shortening. Further,as disclosed in Example 15 below, applicant has also for the first timeshown that Zscan4 expression goes far beyond mere promotion of genomestability. Zscan4 expression in a population of human fibroblast cellshaving trisomy 21 induces the correction of the trisomy 21 abnormalityin approximately 55% of the cells. Accordingly, activating or increasingexpression of Zscan4 can be used to treat aneuploidy in cells, as wellas increase the success rate of in vitro fertilization (IVF) andsuccessful pregnancies in older women by rejuvenating and/or correctingchromosomal abnormalities, such as aneuploidy, in oocyte cells andfertilized oocytes.

Accordingly, the methods of the present disclosure generally relate toincreasing the expression of Zscan4 (e.g., Zscan4 protein expression) inhuman cells to increase telomere length and/or increase genomestability. Various aspects of the present disclosure relate toincreasing telomere length in one or more human cells, treating asubject in need of telomere lengthening, treating a disease or conditionassociated with a telomere abnormality, treating a disease or conditionassociated with a chromosome abnormality, increasing genome stability ofone or more human cells, rejuvenating one or more human cells,rejuvenating a tissue or organ in a subject, and rejuvenating a subjectin need thereof.

In one aspect, the present disclosure relates to a method of increasingtelomere length in one or more human cells, including contacting the oneor more human cells with an agent that increases expression of Zscan4 inthe human cell, where increased expression of Zscan4 induces telomerelengthening in the one or more human cells as compared to one or morecorresponding human cells that are not contacted with the agent.

In another aspect, the present disclosure relates to a method oftreating a subject in need of telomere lengthening, including contactingone or more human cells in the subject with an agent that increasesexpression of Zscan4 in the one or more human cells, where increasedexpression of Zscan4 induces telomere lengthening in the one or morehuman cells.

In another aspect, the present disclosure relates to a method oftreating a subject in need of telomere lengthening, including: i.)isolating one or more human cells in need of telomere lengthening fromthe subject; ii.) contacting the one or more human cells with an agentthat increases expression of Zscan4 in the one or more human cells,where increasing expression of Zscan4 induces telomere lengthening inthe one or more human cells; and iii.) administering the contacted oneor more human cells to the subject.

In another aspect, the present disclosure relates to a method oftreating a disease or condition associated with a telomere abnormality,including administering to a subject in need thereof an agent thatincreases expression of Zscan4 in one or more human cells in thesubject, where increasing expression of Zscan4 induces telomerelengthening in the one or more human cells to treat to treat the totreat the disease or condition associated with a telomere abnormality.

In another aspect, the present disclosure relates to a method oftreating a disease or condition associated with a telomere abnormality,including: i.) isolating one or more human cells from a subjectsuffering from a disease or condition associated with a telomereabnormality; ii.) contacting the one or more human cells with an agentthat increases expression of Zscan4 in the one or more human cells,where increasing expression of Zscan4 induces telomere lengthening inthe one or more human cells; and iii.) administering the contacted oneor more human cells to the subject to treat the disease or conditionassociated with a telomere abnormality.

In another aspect, the present disclosure relates to a method oftreating a disease or condition associated with a chromosomeabnormality, by administering to a subject in need thereof an agent thatincreases expression of Zscan4 in one or more human cells in thesubject, where increasing expression of Zscan4 induces correction of thechromosome abnormality in the one or more human cells to treat thedisease or condition associated with a chromosome abnormality.

In another aspect, the present disclosure relates to a method oftreating a disease or condition associated with a chromosomeabnormality, by: i.) isolating one or more human cells from a subjectsuffering from a disease or condition associated with a chromosomeabnormality; ii.) contacting the one or more human cells with an agentthat increases expression of Zscan4 in the one or more human cells,where increasing expression of Zscan4 induces correction of thechromosome abnormality in the one or more human cells; and iii.)administering the contacted one or more human cells to the subject totreat the disease or condition associated with a chromosome abnormality.

In another aspect, the present disclosure relates to a method oftreating a disease or condition associated with a karyotype abnormality,by administering to a subject in need thereof an agent that increasesexpression of Zscan4 in one or more human cells in the subject, whereincreasing expression of Zscan4 induces correction of the karyotypeabnormality in the one or more human cells to treat the disease orcondition associated with a karyotype abnormality.

In another aspect, the present disclosure relates to a method oftreating a disease or condition associated with a karyotype abnormality,by: i.) isolating one or more human cells from a subject suffering froma disease or condition associated with a karyotype abnormality; ii.)contacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where increasingexpression of Zscan4 induces correction of the karyotype abnormality inthe one or more human cells; and iii.) administering the contacted oneor more human cells to the subject to treat the disease or conditionassociated with a karyotype abnormality.

In another aspect, the present disclosure relates to a method oftreating a cancer, by administering to a subject in need thereof anagent that increases expression of Zscan4 in one or more cancer cells inthe subject, where increasing expression of Zscan4 represses growth ofthe one or more cancer cells, thereby treating the cancer.

In another aspect, the present disclosure relates to a method ofimproving responsiveness to chemotherapy in a cancer patient, byadministering to a subject in need thereof an agent that reducesexpression of endogenous ZSCAN4 in one or more cancer stem cells in thesubject, where reducing expression of endogenous ZSCAN4 reduces oreliminates resistance to one or more chemotherapeutic agents in the oneor more cancer stem cells, thereby improving responsiveness to the oneor more chemotherapeutic agents in the subject.

In another aspect, the present disclosure relates to a method ofincreasing genome stability of one or more human cells, includingcontacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where increasedexpression of Zscan4 increases genome stability in the one or more humancells as compared to one or more corresponding human cells that are notcontacted with the agent.

In another aspect, the present disclosure relates to a method ofincreasing DNA repair capacity of one or more human cells, by contactingthe one or more human cells with an agent that increases expression ofZscan4 in the one or more human cells, where increased expression ofZscan4 increases DNA repair capacity in the one or more human cells ascompared to one or more corresponding human cells that are not contactedwith the agent.

In another aspect, the present disclosure relates to a method ofrejuvenating one or more human cells, including contacting the one ormore human cells with an agent that increases expression of Zscan4 inthe one or more human cells, where increased expression of Zscan4rejuvenates the one or more human cells as compared to one or morecorresponding human cells that are not contacted with the agent.

In another aspect, the present disclosure relates to a method ofproviding resistance to one or more genotoxic agents in one or morehuman cells, by contacting the one or more human cells with an agentthat increases expression of Zscan4 in the one or more human cells,where increased expression of Zscan4 increases resistance to one or moregenotoxic agents in the one or more human cells as compared to one ormore corresponding human cells that are not contacted with the agent.

In another aspect, the present disclosure relates to a method forinducing a human embryonic stem cell-like DNA methylation pattern in oneor more human induced pluripotent stem (iPS) cells, by contacting theone or more human iPS cells with an agent that increases expression ofZscan4 in the one or more human iPS cells, where increased expression ofZscan4 induces a human embryonic stem cell-like DNA methylation patternin the one or more human iPS cells as compared to one or morecorresponding human iPS cells that are not contacted with the agent.

In another aspect, the present disclosure relates to a method ofrejuvenating one or more human oocyte cells, by contacting the one ormore human oocyte cells with an agent that increases expression ofZscan4 in the one or more human oocyte cells, where increased expressionof Zscan4 rejuvenates the one or more human oocyte cells as compared toone or more corresponding human oocyte cells that are not contacted withthe agent.

In another aspect, the present disclosure relates to a method ofincreasing genome stability of one or more human oocyte cells, bycontacting the one or more human oocyte cells with an agent thatincreases expression of Zscan4 in the one or more human oocyte cells,where increased expression of Zscan4 increases genome stability in theone or more human oocyte cells as compared to one or more correspondinghuman oocyte cells that are not contacted with the agent.

In another aspect, the present disclosure relates to a method ofcorrecting one or more karyotype abnormalities in one or more humanoocyte cells, by contacting the one or more human oocyte cells with anagent that increases expression of Zscan4 in the one or more humanoocyte cells, wherein increased expression of Zscan4 induces correctionof the one or more karyotype abnormalities in the one or more humanoocyte cells as compared to one or more corresponding human oocyte cellsthat are not contacted with the agent.

In another aspect, the present disclosure relates to an in vitro methodof increasing genome stability of one or more fertilized human oocytes,by contacting the one or more fertilized human oocytes with an agentthat increases expression of Zscan4 in the one or more fertilized humanoocytes, where increased expression of Zscan4 increases genome stabilityin the one or more fertilized human oocytes as compared to one or morecorresponding fertilized human oocytes that are not contacted with theagent.

In another aspect, the present disclosure relates to an in vitro methodof correcting one or more karyotype abnormalities in one or morefertilized human oocytes, by contacting the one or more fertilized humanoocytes with an agent that increases expression of Zscan4 in the one ormore fertilized human oocytes, where increased expression of Zscan4induces correction of the one or more karyotype abnormalities in the oneor more fertilized human oocytes as compared to one or morecorresponding fertilized human oocytes that are not contacted with theagent.

In another aspect, the present disclosure relates to a method oftreating a disease or condition associated with a telomere abnormality,including: i.) isolating human bone marrow cells from a subjectsuffering from a disease or condition associated with a telomereabnormality; ii.) contacting the human bone marrow cells with an agentthat increases expression of Zscan4 in the human bone marrow cells,where increasing expression of Zscan4 induces telomere lengthening inthe human bone marrow cells; and iii.) engrafting the contacted humanbone marrow cells into the subject to treat the disease or conditionassociated with a telomere abnormality.

In another aspect, the present disclosure relates to a method oftreating a disease or condition associated with a chromosomeabnormality, including: i.) isolating human bone marrow cells from asubject suffering from a disease or condition associated with achromosome abnormality; ii.) contacting the human bone marrow cells withan agent that increases expression of Zscan4 in the human bone marrowcells, where increasing expression of Zscan4 induces correction of thechromosome abnormality in the human bone marrow cells; and iii.)engrafting the contacted human bone marrow cells into the subject totreat the disease or condition associated with a chromosome abnormality.

In another aspect, the present disclosure relates to a method ofrejuvenating a tissue or organ in a subject, including administering toa subject in need thereof an agent that increases expression of Zscan4in the tissue or organ, where increasing expression of Zscan4rejuvenates the tissue or organ.

In another aspect, the present disclosure relates to a method ofrejuvenating a subject in need thereof, including administering to thesubject an agent that increases expression of Zscan4, where increasingexpression of Zscan4 rejuvenates the subject.

In another aspect, the present disclosure relates to a method ofextending lifespan of one or more human cells, by contacting the one ormore human cells with an agent that increases expression of Zscan4 inone or more human cells in the subject, where increasing expression ofZscan4 extends the lifespan of the one or more human cells as comparedto one or more corresponding human cells that are not contacted with theagent.

In another aspect, the present disclosure relates to a method ofextending lifespan of a tissue or organ in a subject, by administeringto a subject in need thereof an agent that increases expression ofZscan4 in the tissue or organ, where increasing expression of Zscan4extends the lifespan of the tissue or organ.

In another aspect, the present disclosure relates to a method ofextending lifespan of a subject, by administering to a subject in needthereof an agent that increases expression of Zscan4 in one or morehuman cells in the subject, where increasing expression of Zscan4extends the lifespan of the one or more human cells, thereby extendingthe lifespan of the subject.

In another aspect, the present disclosure relates to a method ofextending lifespan of a subject, by: i. isolating one or more humancells from the subject; ii. contacting the one or more human cells withan agent that increases expression of Zscan4 in the one or more humancells, where increasing expression of Zscan4 extends the lifespan of theone or more human cells; and iii. administering the contacted one ormore human cells to the subject to extend the lifespan of the subject.

In another aspect, the present disclosure relates to a method fordetermining one or more Zscan4-induced effects in one or more humancells, by: i. contacting the one or more human cells with an agent thatincreases expression of Zscan4 in one or more human cells; ii. measuringexpression levels of SERPINB4, DNMT3L, and/or DUX4 in the one or morehuman cells; and iii. comparing the expression levels of SERPINB4,DNMT3L, and/or DUX4 in the one or more human cells to the expressionlevels of SERPINB4, DNMT3L, and/or DUX4 in one or more correspondinghuman cells that are not contacted with the agent, where an increase inthe expression levels of SERPINB4, DNMT3L, and/or DUX4 in the one ormore human cells indicates the presence of one or more Zscan4-inducedeffects in the one or more human cell.

Zscan4

Zinc finger and SCAN domain containing 4 (Zscan4) genes represent agroup of genes that have previously been identified as exhibiting2-cell-specific expression and ES cell-specific expression (PCTPublication No. WO 2008/118957). The Zscan4 gene was identified byexpression profiling of all pre-implantation stages of mouse embryosusing a large-scale cDNA sequencing project (Ko et al., Development 127:1737-1749, 2000; Sharov et al., PLoS Biol 1:E74, 2003) and DNAmicroarray analysis (Hamatani et al, Dev Cell 6:117-131, 2004). In mice,the term “Zscan4” refers to a collection of genes including threepseudogenes (Zscan4-ps1, Zscan4-ps2 and Zscan4-ps3) and six expressedgenes (Zscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e and Zscan4f). Amongthe six paralogs, the open reading frames of Zscan4c, Zscan4d, andZscan4f encode a SCAN domain, predicted to mediate protein-proteininteractions, as well as four zinc finger domains, suggesting theirpotential role as transcription factors. In contrast to mice, the humangenome contains only one copy of Zscan4. Zscan4 may refer to Zscan4polypeptides and Zscan4 may refer to polynucleotides encoding the Zscan4polypeptides.

It has recently been shown that Zscan4 (Zinc finger and scandomain-containing protein 4), which, in mice, is expressed specificallyin 2-cell stage embryos and ES cells (Falco et al., Dev Biol307:539-550, 2007), is required for the maintenance of genome stabilityand normal karyotype in ES cells (Zalzman et al., Nature 464:858-863,2010). Although only a small fraction (˜1% to ˜5%) of undifferentiatedES cells express Zscan4 at a given time (Falco et al., Dev Biol307:539-550, 2007), essentially all of the ES cells in culture undergothe transient Zscan4+ state within 9 passages (Zalzman et al., Nature464:858-863, 2010). Upon short hairpin RNA (shRNA)-mediated repressionof Zscan4, after about 8 passages ES cells undergo massive karyotypedeterioration. Prior studies have also shown that the Zscan4+ state ofmouse ES cells is associated with telomere extension (Zalzman et al.,Nature 464:858-863, 2010). Although ES cells have the best capacity tomaintain their genome integrity in culture, it is also widely recognizedthat even ES cells, in long-term culture, gradually lose theirdevelopmental potency. A telomere may refer to the end of a eukaryoticchromosome, a specialized structure involved in the replication andstability of the chromosome. Telomeres contain many repeats of a shortDNA sequence in a specific orientation. Telomere functions includeprotecting the ends of the chromosome so that chromosomes do not end upjoined together, and allowing replication of the extreme ends of thechromosomes (by telomerase). The number of repeats of telomeric DNA atthe end of a chromosome decreases with age.

It has also been shown previously that forced expression of mouse Zscan4in mouse ES cells for three days increases the average length oftelomeres from the standard length of approximately 40 kb toapproximately 66 kb (Zalzman et al., 2010). This indicates that Zscan4alone can efficiently and rapidly increase telomere length. However, itis unknown whether Zscan4 can increase the length of telomeres innon-embryonic human cells, such as adult stem cells and somatic cells.

Human Cells

Certain aspects of the present disclosure relate to increasing telomerelength in one or more human cells, including without limitation, humanadult cells, by utilizing an agent that increases Zscan4 expression(e.g., Zscan4 protein expression) in the one or more human cells. Incertain embodiments, the one or more human cells are in a subject inneed of telomere lengthening, or suffering or diagnosed with a diseaseor condition associated with a telomere abnormality.

Various human cells find use in the methods described herein. Asdisclosed herein, the term “human cell(s)” refers to any cell(s) foundthroughout the human body during and after embryonic development, suchas human embryonic cells, stem cells, pluripotent cells, differentiatedcells, mature cells, somatic cells, and adult cells. In someembodiments, human cells of the present disclosure are human adultcells. As disclosed herein, the term “human adult cell(s)” refers to anycell(s) found throughout the human body after embryonic development(i.e., non-embryonic cells). Human cells of the present disclosureinclude, without limitation, sperm cells, oocyte cells, fertilizedoocytes (i.e., zygotes), embryonic cells, mature cells, differentiatedcells, somatic cells, progenitor cells, embryonic stem (ES) cells,induced pluripotent stem (iPS) cells, adult stem cells, somatic stemcells, and tissue stem cells. Adult stem cells, which are also known assomatic stem cells or tissue stem cells, may refer to undifferentiatedcells, found throughout the body after embryonic development, whichmultiply by cell division to replenish dying cells and regeneratedamaged tissues. Progenitor cells may refer to oligopotent or unipotentcells that differentiate into a specific type of cell or cell lineage.Progenitor cells are similar to stem cells but are more differentiatedand exhibit limited self-renewal. Exemplary adult stem cells, tissuestem cells, and/or progenitor cells may include, without limitation,hematopoietic stem cells, mesenchymal stem cells, adipose stem cells,neuronal stem cells, intestinal stem cells, skin stem cells, and germcells (such as, sperm cells and oocytes).

Human cells may also include, without limitation, somatic cells, maturecells, and differentiated cells. Somatic cells may refer to any cell ofthe body, including, without limitation, germ cells, tissue stem cells,progenitor cells, induced pluripotent stem (iPS) cells, anddifferentiated cells. Exemplary somatic cells, mature cells, and/ordifferentiated cells may include, without limitation, epidermal cells,fibroblasts, lymphocytes, hepatocytes, epithelial cells, myocytes,chondrocytes, osteocytes, adipocytes, cardiomyocytes, pancreatic βcells, keratinocytes, erythrocytes, peripheral blood cells, bone marrowcells, neurocytes, astrocytes, and germ cells. Germ cells may refer tothe cells that give rise to the gametes (i.e., eggs and sperm) oforganisms that reproduce sexually. In certain embodiments, germ cellsinclude, without limitation, oocytes, and sperm cells. In someembodiment, somatic cells, mature cells, and/or differentiated cells ofthe present disclosure also include, without limitation, preimplantationembryos.

Agents that Increase Expression of Zscan4

Certain aspects of the present disclosure relate to utilizing an agentthat increases Zscan4 expression (e.g., Zscan4 protein expression) inhuman cells to increase telomere length in the human cells. An agent mayrefer to any nucleic acid molecule, protein, compound, small molecule,organic compound, inorganic compound, or other molecule of interest. Insome embodiments, the agent is any agent that increases expression ofZscan4 either by directly interacting with the endogenous Zscan4 gene(including any upstream or downstream regulatory sequences) or byinteracting with genes and/or proteins that lead to the induction ofZscan4 expression. In some embodiments, the agent can be a nucleic acidmolecule encoding Zscan4 including, without limitation, a synthetic mRNAand an expression vector including, without limitation, a viral vectorsuch as Sendai virus vectors. In other embodiments, the agent can be apolypeptide containing a Zscan4 protein or a functional portion thereofsuch as Zscan4-AC. In some embodiments, the agent can be a retinoid, oran agent that induces oxidative stress.

In some embodiments, an agent of the present disclosure that increasesZscan4 expression (e.g., Zscan4 protein expression) in human cellstransiently increases Zscan4 expression. For example, an agent of thepresent disclosure that increases Zscan4 expression in human cells mayincrease Zscan4 expression for about 1 hour to about 23 hours (e.g., forabout 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about10 hours, 11 hours, about 12 hours, about 13 hours, about 14 hours,about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, or about 23hours); or about 1 day to about 10 days (e.g., about 1 day, about 1.25days, about 1.5 days, about 1.75 days, about 2 days, about 2.25 days,about 2.5 days, about 2.75 days, about 3 days, about 3.25 days, about3.5 days, about 3.75 days, about 4 days, about 4.25 days, about 4.5days, about 4.75 days, about 5 days, about 6.25 days, about 6.5 days,about 6.75 days, about 7 days, about 7.25 days, about 7.5 days, about7.75 days, about 8 days, about 8.25 days, about 8.5 days, about 8.75days, about 9 days, about 9.25 days, about 9.5 days, about 9.75 days, orabout 10 days).

In some embodiments, the disclosed beneficial effects of increasedZscan4 expression (e.g., Zscan4 protein expression) in human cells maybe enhanced by repeated transient increases in Zscan4 expression.Accordingly, in certain embodiments, an agent of the present disclosurethat increases Zscan4 expression (e.g., Zscan4 protein expression) inhuman cells may be used to repeatedly increase Zscan4 expression inhuman cells at an interval of every 4 hours, every 8 hours, every 12hours, every 16 hours, every 24 hours, every 32 hours, every 40 hours,every 48 hours, every three days, every four days, every five days,every six days, every week, every two weeks, every three weeks, everyfour weeks, every month, every two months, every three months, everyfour months, every six months, every seven months, every eight months,every nine months, every 10 months, every 11 months, every year, everytwo years, every three years, every four years, every five years, everysix years, every seven years, every eight years, every nine years, every10 years, every 11 years, every 12 years, every 13 years, every 14years, every 15 years, every 16 years, every 17 years, every 18 years,every 19 years, every 20 years, every 21 years, every 22 years, every 23years, every 24 years, every 25 years, every 26 years, every 27 years,every 28 years, every 29 years, every 30 years, every 35 years, every 40years, every 45 years, or every 50 years.

As disclosed herein, human cells do not generally express ZSCAN proteinin any significant amount. As such, agents of the present disclosurethat increases Zscan4 expression increase Zscan4 protein expression intreated cells. In some embodiments, treating a human cell with an agentof the present disclosure that increases Zscan4 expression may result inat least a 1.5 fold increase to at least a 1,000,000 fold increase inZscan4 protein expression.

Accordingly, in certain embodiments, an agent of the present disclosurethat increases Zscan4 expression in human cells, increases Zscan4protein expression by at least 1.5 fold, at least 1.6 fold, at least 1.7fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least2.1 fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, atleast 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, atleast 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold,at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0fold, at least 9.5 fold, at least 10 fold, at least 100 fold, at least200 fold, at least 300 fold, at least 400 fold, at least 500 fold, atleast 600 fold, at least 700 fold, at least 800 fold, at least 900 fold,at least 1,000 fold, at least 2,000 fold, at least 3,000 fold, at least4,000 fold, at least 5,000 fold, at least 6,000 fold, at least 7,000fold, at least 8,000 fold, at least 9,000 fold, at least 10,000 fold, atleast 25,000 fold, at least 50,000 fold, at least 75,000 fold, at least100,000 fold, at least 125,000 fold, at least 150,000 fold, at least175,000 fold, at least 200,000 fold, at least 225,000 fold, at least250,000 fold, at least 275,000 fold, at least 300,000 fold, at least325,000 fold, at least 350,000 fold, at least 375,000 fold, at least400,000 fold, at least 425,000 fold, at least 450,000 fold, at least475,000 fold, at least 500,000 fold, at least 525,000 fold, at least550,000 fold, at least 575,000 fold, at least 600,000 fold, at least625,000 fold, at least 650,000 fold, at least 675,000 fold, at least700,000 fold, at least 725,000 fold, at least 750,000 fold, at least775,000 fold, at least 800,000 fold, at least 825,000 fold, at least850,000 fold, at least 875,000 fold, at least 900,000 fold, at least925,000 fold, at least 950,000 fold, at least 975,000 fold, or at least1,000,000 fold, for example, relative to Zscan4 protein expression in ahuman cell that has not been contacted with the agent.

Any method known in the art and disclosed herein for determining Zscan4protein expression in a cell, or for quantifying the number of proteins(i.e., protein stoichiometry) per cell may be used. In some embodiments,not all cells in a cell population or subject treated with the agentwill be affected by the agent. For example, in embodiments where theagent is a viral vector expressing Zscan4, the viral vector may notinfect every cell in a treated cell population or subject. As such, a“fold increase” in Zscan4 protein expression as used herein refers tothe average increase in Zscan4 protein expression in the cells in atreated cell population or subject that are affected by the agent. Forexample in embodiments where the agent is a viral vector, a foldincrease in Zscan4 protein expression may refer to the average increasein Zscan4 protein expression in infected cells of a treated cellpopulation or subject.

Zscan4 Polynucleotides

In some embodiments, an agent of the present disclosure that increasesexpression of Zscan4 is a nucleic acid molecule including a nucleic acidsequence encoding a Zscan4 protein. A polynucleotide may refer to anucleic acid sequence (such as a linear sequence) of any length.Therefore, a polynucleotide includes oligonucleotides, and also genesequences found in chromosomes. An oligonucleotide is a plurality ofjoined nucleotides joined by native phosphodiester bonds. Anoligonucleotide is a polynucleotide of between 6 and 300 nucleotides inlength. An oligonucleotide analog refers to moieties that functionsimilarly to oligonucleotides but have non-naturally occurring portions.For example, oligonucleotide analogs can contain non-naturally occurringportions, such as altered sugar moieties or inter-sugar linkages, suchas a phosphorothioate oligodeoxynucleotide. Functional analogs ofnaturally occurring polynucleotides can bind to RNA or DNA, and includepeptide nucleic acid (PNA) molecules.

Nucleic acid molecules encoding a Zscan4 polypeptide are termed Zscan4polynucleotides or nucleic acid molecules. These polynucleotides includeDNA, cDNA and RNA sequences, such as mRNA sequences, which encode aZscan4. It is understood that all polynucleotides encoding a Zscan4polypeptide are also included herein, as long as they encode apolypeptide with a recognized Zscan4 activity, such as the ability tomodulate genome stability or telomere length. Genome stability may referto the ability of a cell to faithfully replicate DNA and maintainintegrity of the DNA replication machinery. Long telomeres are thoughtto provide a buffer against cellular senescence and be generallyindicative of genome stability and overall cell health. Chromosomestability (e.g., few mutations, no chromosomal rearrangements or changein number) is also associated with genome stability. A loss of genomestability is associated with cancer, neurological disorders andpremature aging. Signs of genome instability include elevated mutationrates, gross chromosomal rearrangements, alterations in chromosomenumber, and shortening of telomeres.

Zscan4 nucleic acid sequences have been previously described in the art(see, for example, WO 2008/118957, the disclosure of which is hereinincorporated by reference; Falco et al., Dev. Biol. 307(2):539-550,2007; and Carter et al., Gene Expr. Patterns. 8(3):181-198, 2008).Zscan4 nucleic acids may include, without limitation, any one of a groupof mouse Zscan4 genes exhibiting 2-cell embryonic stage- or EScell-specific expression (including Zscan4a, Zscan4b, Zscan4c, Zscan4d,Zscan4e and Zscan4f), the human ortholog ZSCAN4, or any other speciesortholog of Zscan4.

As disclosed herein, the nucleotide sequence of the mouse Zscan4a geneis set forth in SEQ ID NO: 1, the nucleotide sequence of the mouseZscan4b gene is set forth in SEQ ID NO: 2, the nucleotide sequence ofthe mouse Zscan4c gene is set forth in SEQ ID NO: 3, the nucleotidesequence of the mouse Zscan4d gene is set forth in SEQ ID NO: 4, thenucleotide sequence of the mouse Zscan4e gene is set forth in SEQ ID NO:5, and the nucleotide sequence of the mouse Zscan4f gene is set forth inSEQ ID NO: 6. Additionally, the nucleotide sequence of the human ZSCAN4gene is set forth in SEQ ID NO: 7.

Zscan4 nucleic acid sequences from other species are publicallyavailable, including dog Zscan4 (GenBank Accession Nos. XM.sub.—541370.2and XM.sub.—848557.1; SEQ ID NO: 8); cow Zscan4 (GenBank Accession No.XM.sub.—001789250.1; SEQ ID NO: 9); horse Zscan4 (GenBank Accession No.XM.sub.—001493944.1; SEQ ID NO: 10); gorilla Zscan4 (nucleotide sequenceof UniProt Accession No. A1YEQ9; SEQ ID NO: 21); bonobo Zscan4(nucleotide sequence of UniProt Accession No. A1YFX5; SEQ ID NO: 22);Bornean orangutan Zscan4 (nucleotide sequence of UniProt Accession No.A2T7G6; SEQ ID NO: 23); Sumatran orangutan (nucleotide sequence ofUniProt Accession No. H2POE3; SEQ ID NO: 24); panda Zscan4 (nucleotidesequence of UniProt Accession No. G1LE29; SEQ ID NO: 25); pig Zscan4(nucleotide sequence of UniProt Accession No. F1SCQ2; SEQ ID NO: 26);Northern white-cheeked gibbon Zscan4 (nucleotide sequence of UniProtAccession No. G1RJD4; SEQ ID NO: 27); Rhesus macaque Zscan4 (nucleotidesequence of UniProt Accession No. F7GH55; SEQ ID NO: 28); guinea pigZscan4 (nucleotide sequence of UniProt Accession No. HOV5E8; SEQ ID NO:29); and Thirteen-lined ground squirrel (nucleotide sequence of UniProtAccession No. I3N7T3; SEQ ID NO: 30). Each of the above-listed GenBankAccession numbers is herein incorporated by reference as it appears inthe GenBank database on Aug. 11, 2009. Each of the above-listed UniProtAccession numbers is herein incorporated by reference as it appears inthe UniProt database on Mar. 15, 2013.

In a specific example, Zscan4 is mouse Zscan4c or human ZSCAN4. Zscan4nucleic acids may also include, without limitation, Zscan4 nucleicacids, or homologs thereof, that encode Zscan4 polypeptides that arecapable of increasing genome stability and/or increasing telomerelength.

Fragments and variants of Zscan4 polynucleotides can readily be preparedby one of skill in the art using molecular techniques. In someembodiments, a fragment of a Zscan4 polynucleotide includes at least250, at least 500, at least 750, at least 1000, at least 1500, or atleast 2000 consecutive nucleic acids of a Zscan4 polynucleotide. In someembodiments, a fragment of Zscan4 is a fragment that confers a functionof Zscan4 when expressed in a cell of interest, such as, but not limitedto, increasing genome stability and/or increasing telomere length.

Minor modifications of the Zscan4 polynucleotide sequences may result inexpression of peptides which have substantially equivalent activity ascompared to the unmodified counterpart polynucleotides described herein.Such modifications may be deliberate, as by site-directed mutagenesis,or may be spontaneous. All of the polynucleotides produced by thesemodifications are included herein.

Zscan4 polynucleotides may include recombinant DNA which is incorporatedinto a vector; into an autonomously replicating plasmid or virus; orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA) independent of other sequences. Thenucleotides can be ribonucleotides, deoxyribonucleotides, or modifiedforms of either nucleotide. The term includes single- anddouble-stranded forms of DNA. A recombinant nucleic acid or polypeptideis one that has a sequence that is not naturally occurring or has asequence that is made by an artificial combination of two otherwiseseparated segments of sequence. This artificial combination is oftenaccomplished by chemical synthesis or by the artificial manipulation ofisolated segments of nucleic acids, for example, by genetic engineeringtechniques.

In some embodiments, a degenerative variant of any of the Zscan4polynucleotides described herein may be used in the methods of thepresent disclosure. A degenerative variant may refer to a polynucleotideencoding a polypeptide, such as a Zscan4 polypeptide, that includes asequence that is degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included aslong as the amino acid sequence of the polypeptide encoded by thenucleotide sequence is unchanged.

A Zscan4 coding sequence may be operably linked to a heterologouspromoter to direct transcription of the Zscan4 coding nucleic acidsequence. A promoter may refer to nucleic acid control sequences whichdirect transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription. A promoteralso optionally includes distal enhancer or repressor elements. Aconstitutive promoter is a promoter that is continuously active and isnot subject to regulation by external signals or molecules. In contrast,the activity of an inducible promoter is regulated by an external signalor molecule (for example, a transcription factor). A first nucleic acidsequence is operably linked to a second nucleic acid sequence when thefirst nucleic acid sequence is placed in a functional relationship withthe second nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked nucleicacid sequences are contiguous and where necessary to join two proteincoding regions, in the same reading frame. A heterologous polypeptide orpolynucleotide refers to a polypeptide or polynucleotide derived from adifferent source or species. A promoter includes necessary nucleic acidsequences near the start site of transcription, such as, in the case ofa polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription. In one example, the promoter is a constitutive promoter,such as the CAG-promoter (Niwa et al., Gene 108(2):193-9, 1991), or thephosphoglycerate kinase (PGK)-promoter. In some embodiments, thepromoter is an inducible promoter such as a tetracycline-induciblepromoter (Masui et al., Nucleic Acids Res. 33:e43, 2005). Otherexemplary promoters that can be used to drive Zscan4 expression includebut are not limited to: lac system, the trp system, the tac system, thetrc system, major operator and promoter regions of phage lambda, thecontrol region of fd coat protein, the early and late promoters of SV40,promoters derived from polyoma, adenovirus, retrovirus, baculovirus andsimian virus, the promoter for 3-phosphoglycerate kinase, the promotersof yeast acid phosphatase, and the promoter of the yeast alpha-matingfactors. In some embodiments, a native Zscan4 promoter is used. Zscan4polynucleotides of the present disclosure may be under the control of aconstitutive promoter, an inducible promoter, or any other suitablepromoter described herein or other suitable promoter that will bereadily recognized by one skilled in the art.

The identity/similarity between two or more nucleic acid sequences, ortwo or more amino acid sequences, is expressed in terms of the identityor similarity between the sequences. Sequence identity can be measuredin terms of percentage identity; the higher the percentage, the moreidentical the sequences are. Sequence similarity can be measured interms of percentage similarity (which takes into account conservativeamino acid substitutions); the higher the percentage, the more similarthe sequences are. Homologs or orthologs of nucleic acid or amino acidsequences possess a relatively high degree of sequenceidentity/similarity when aligned using standard methods. This homologyis more significant when the orthologous proteins or cDNAs are derivedfrom species which are more closely related (such as human and mousesequences), compared to species more distantly related (such as humanand C. elegans sequences).

The terms “identical” or percent “identity,” in the context of two ormore sequences (e.g., nucleic acid sequences or amino acid sequences),may refer to two or more sequences or subsequences that are the same.Two sequences are substantially identical if two sequences have aspecified percentage of amino acid residues or nucleotides that are thesame (i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specifiedregion, or, when not specified, over the entire sequence), when comparedand aligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. When comparing two sequences foridentity, it is not necessary that the sequences be contiguous, but anygap would carry with it a penalty that would reduce the overall percentidentity. For blastp, the default parameters are Gap opening penalty=11and Gap extension penalty=1. For blastn, the default parameters are Gapopening penalty=5 and Gap extension penalty=2.

A comparison window may include reference to a segment of any one of thenumber of contiguous positions including, but not limited to from 20 to600, usually about 50 to about 200, more usually about 100 to about 150in which a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequences for comparison are well knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(1981), by the homology alignment algorithm of Needleman and Wunsch(1970) J Mol Biol 48(3):443-453, by the search for similarity method ofPearson and Lipman (1988) Proc Natl Acad Sci USA 85(8):2444-2448, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection [see, e.g., Brent et al., (2003) Current Protocols inMolecular Biology, John Wiley & Sons, Inc. (Ringbou Ed)].

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nucleic AcidsRes 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol215(3)-403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. For aminoacid sequences, the BLASTP program uses as defaults a word length of 3,and expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoffand Henikoff, (1992) Proc Natl Acad Sci USA 89(22):10915-10919]alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. For nucleotide sequences, the BLASTN program uses asdefaults a word length (W) of 11, an expectation (E) or 10, M=5, N=−4,and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, (1993)Proc Natl Acad Sci USA 90(12):5873-5877). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions. Nucleic acid sequences that do not show a high degree ofidentity may nevertheless encode identical or similar (conserved) aminoacid sequences, due to the degeneracy of the genetic code. Changes in anucleic acid sequence can be made using this degeneracy to producemultiple nucleic acid molecules that all encode substantially the sameprotein. An alternative (and not necessarily cumulative) indication thattwo nucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.

Moreover, one indication that two polypeptides are substantiallyidentical is that the first polypeptide is immunologicallycross-reactive with antibodies raised against the second polypeptide.Thus, a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions.

Various aspects of the present disclosure relate to isolated entities,such as isolated nucleic acids or synthetic mRNA molecules. An isolatednucleic acid has been substantially separated or purified away fromother nucleic acid sequences and from the cell of the organism in whichthe nucleic acid naturally occurs, i.e., other chromosomal andextrachromosomal DNA and RNA. The term “isolated” thus encompassesnucleic acids purified by standard nucleic acid purification methods.The term also embraces nucleic acids prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.Similarly, isolated proteins have been substantially separated orpurified from other proteins of the cells of an organism in which theprotein naturally occurs, and encompasses proteins prepared byrecombination expression in a host cell as well as chemicallysynthesized proteins. Similarly, isolated cells have been substantiallyseparated away from other cell types.

Accordingly, in certain embodiments, the polynucleotides of the presentdisclosure include sequences that are degenerate as a result of thegenetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included as long as the amino acid sequence of the Zscan4polypeptide encoded by the nucleotide sequence is functionallyunchanged. A Zscan4 polynucleotide encodes a Zscan4 polypeptide, asdisclosed herein. Exemplary polynucleotide sequences encoding Zscan4polypeptides may include, for example, the nucleotide sequence from anyone of SEQ ID NOs: 1-10 and 21-30. Further, non-human homologs of humanZSCAN4 may be used to increase Zscan4 expression in a human subject inaccordance with any of the methods of the present disclosure, asexpression of such non-human Zscan4 homologs is transient, and as suchwould not lead to an adverse immunogenic response in the human subject.

In some embodiments, the Zscan4 polynucleotide encoding a Zscan4polypeptide is a human ZSCAN4 polynucleotide or a homolog thereof. Insome embodiments, the Zscan4 polynucleotide encoding a Zscan4polypeptide is a mouse Zscan4 polynucleotide or a homolog thereof. Insome embodiments, the Zscan4 polynucleotide encoding a Zscan4polypeptide is a Zscan4a polynucleotide, a Zscan4b polynucleotide, aZscan4c polynucleotide, a Zscan4d polynucleotide, a Zscan4epolynucleotide, or a Zscan4f polynucleotide. In some embodiments, theZscan4 polynucleotide encoding a Zscan4 polypeptide is a dog Zscan4polynucleotide, a cow Zscan4 polynucleotide, a horse Zscan4polynucleotide, or a homolog thereof. In some embodiments, the Zscan4polynucleotide encoding a Zscan4 polypeptide includes a nucleotidesequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto the nucleotide sequence from any one of SEQ ID NO: 1-10 and 21-30.

In certain embodiments, the Zscan4 polynucleotide encoding a Zscan4polypeptide is a human ZSCAN4 polynucleotide or homolog thereof. In someembodiments, the Zscan4 polynucleotide encoding a Zscan4 polypeptideincludes a nucleotide sequence that is at least 70%, at least 75%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 7.

Methods of Introducing Zscan4 Polynucleotides into Human Cells

In some embodiments, Zscan4 polynucleotides are introduced into humancells. Introducing a nucleic acid molecule or a protein into a cellencompasses any means of delivering the nucleic acid molecule or proteininto the cell. For example, nucleic acid molecules can be transfected,transduced or electroporated into a cell. Delivery of proteins intocells can be achieved, for example, by fusing the protein to acell-penetrating peptide, such as a peptide with a protein transductiondomain (e.g., HIV-1 Tat), or a poly-arginine peptide tag (Fuchs andRaines, Protein Science 14:1538-1544, 2005). Protein transductiondomains may refer to small cationic peptides that facilitate entry oflarger molecules (proteins, nucleic acid molecules etc.) into a cell bya mechanism that is independent of classical endocytosis. Apoly-arginine peptide tag may refer to a short peptide (generally 7 to11 residues) comprised of arginine residues that facilitates delivery oflarger molecules (such as proteins and nucleic acid molecules) intocells (see, for example, Fuchs and Raines, Protein Science 14:1538-1544,2005).

Introduction of Zscan4 polynucleotides into human cells may involveusing a viral vector (such as integrating or non-integrating viralvectors) or a plasmid vector, delivery of mRNA molecules encoding theZscan4 polynucleotides, or direct delivery of the Zscan4 proteins. Eachof these methods has been described in the art and is therefore withinthe capabilities of one of skill in the art. A brief summary of eachmethod that can be used to deliver Zscan4 to a human cell is providedherein. A vector may refer to a nucleic acid molecule as introduced intoa host cell, thereby producing a transformed host cell. A vector mayinclude nucleic acid sequences that permit it to replicate in a hostcell, such as an origin of replication (DNA sequences that participatein initiating DNA synthesis). For example, an expression vector containsthe necessary regulatory sequences to allow transcription andtranslation of inserted gene or genes. A vector may also include one ormore selectable marker genes and other genetic elements known in theart. Vectors may include, for example, virus vectors and plasmidvectors.

Zscan4 Promoter Sequences and Expression Vectors

An expression vector including a Zscan4 promoter sequence operablylinked to a nucleic acid sequence encoding a heterologous polypeptide(such as a reporter gene) can be used to identify cells that expressZscan4. Methods of detecting expression of the reporter gene varydepending upon the type of reporter gene and are well known in the art.For example, when a fluorescent reporter is used, detection ofexpression can be achieved by FACS or fluorescence microscopy.Identification of human cells expressing Zscan4 can be achieved withalternative methods, including, but not limited to, using antibodiesspecific for Zscan4 or by in situ hybridization.

In some embodiments, a heterologous nucleic acid sequence (such as areporter molecule) is expressed under the control of a Zscan4 promoter(for example in a vector). A Zscan4 promoter may be a promoter sequencethat regulates the expression of an endogenous Zscan4 polynucleotidedescribed herein. Identification of Zscan4 promoters is well within thecapabilities of one skilled in the art and in view of the presentdisclosure. Other expression control sequences, including appropriateenhancers, transcription terminators, a start codon (i.e., ATG) in frontof a protein-encoding gene, splicing signals for introns, and stopcodons can be included with the Zscan4 promoter in an expression vector.Generally the promoter includes at least a minimal sequence sufficientto direct transcription of a heterologous nucleic acid sequence. In someembodiments, the heterologous nucleic acid sequence encodes a reportermolecule.

In some embodiments, a heterologous nucleic acid sequence (such as areporter molecule) is incorporated into a subject's genomic DNA, such asby homologous recombination. For example, the coding sequence for GFPcould be inserted into the coding region of Zscan4, or could replace thecoding region of Zscan4, such that GFP is expressed in the same manneras endogenous Zscan4. Gene “knock-in” methods by homologousrecombination are well known in the art.

The heterologous protein encoded by the heterologous nucleic acidsequence is typically a reporter molecule, such as a marker, an enzyme,a fluorescent protein, a polypeptide that confers antibiotic resistanceto the cell or an antigen that can be identified using conventionalmolecular biology procedures. Reporter molecules can be used to identifya cell, or a population of cells, of interest, such as human cells thathave been contacted with an agent that increases Zscan4 expression in ahuman cell. In some embodiments, the heterologous protein is afluorescent marker (such as a green fluorescent protein, or a variantthereof, e.g. Emerald (Invitrogen, Carlsbad, Calif.)) an antigenicmarker (such as human growth hormone, human insulin, human HLAantigens); a cell-surface marker (such as CD4, or any cell surfacereceptor); or an enzymatic marker (such as lacZ, alkaline phosphatase).Expression of the reporter gene indicates the cell expresses Zscan4.Methods of detecting expression of the reporter gene vary depending uponthe type of reporter gene and are well known in the art. For example,when a fluorescent reporter is used, detection of expression can beachieved by FACS or fluorescence microscopy.

Expression vectors typically contain an origin of replication as well asspecific genes which allow phenotypic selection of the transformedcells, such as an antibiotic resistance gene. Vectors suitable for useherein are well known in the art, including viral vectors and plasmidvectors (such as those described herein). In some embodiments, anenhancer is located upstream of the Zscan4 promoter, but enhancerelements can generally be located anywhere on the vector and still havean enhancing effect. However, the amount of increased activity willgenerally diminish with distance. Additionally, two or more copies of anenhancer sequence can be operably linked one after the other to producean even greater increase in promoter activity.

Expression vectors including a Zscan4 promoter can be used to transfecthost cells, such as, for example, human cells. Biologically functionalviral and plasmid DNA vectors capable of expression and replication in ahost are known in the art, and can be used to transfect any cell ofinterest. A host cell may refer to cells in which a vector can bepropagated and its DNA expressed. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

A transfected cell may refer to a host cell into which (or into anancestor of which) has been introduced a nucleic acid molecule (e.g.,DNA molecule), such as a DNA molecule including a Zscan4 promoterelement. The process of transfecting or transfection may refer to theprocess of introducing a nucleic acid into a cell or tissue.Transfection can be achieved by any one of a number of methods, such as,but not limited to, liposomal-mediated transfection, electroporation andinjection. Transfection of a host cell with a recombinant nucleic acidmolecule may be carried out by conventional techniques as are well knownto those skilled in the art. Transfection may include liposomal-mediatedtransfection, electroporation, injection or any other suitable techniquefor introducing a nucleic acid molecule into a cell.

Viral Vectors

In some embodiments, the vectors used in the methods of the presentdisclosure are viral vectors. Various viral vectors are known in the artand are described herein.

Paramyxoviruses may be used in the methods of the present disclosure. Aparamyxovirus vector may include, without limitation, a vector (orcarrier) that is derived from the Paramyxovirus and that is used forgene transfer, such as a Zscan4 polynucleotide, to host cells, such ashuman cells. The paramyxovirus vector may be ribonucleoprotein (RNP) ora virus particle having infectivity. Infectivity may refer to theability of a paramyxovirus vector to transfer, through its cell adhesionand membrane fusion abilities, a gene contained in the vector to cellsto which the vector is adhered. The paramyxovirus vector may havereplication ability or may be a defective vector without the replicationability. Replication ability may refer to the ability of paramyxovirusvectors to replicate and produce infective virus particles in host cellsinfected with the virus vectors. (See e.g. US 2004/0005296).

A paramyxovirus is a virus of the Paramyxoviridae family or a derivativethereof. Paramyxoviruses may include, without limitation, virusesbelonging to the Paramyxoviridae such as Sendai virus, Newcastle diseasevirus, Mumps virus, Measles virus, Respiratory syncytial virus,rinderpest virus, distemper virus, simian parainfluenza virus (SV5), andtype I, II, and III human parainfluenza virus. A viral vector usedherein may be based on a virus of the genus Paramyxovirus or aderivative thereof. A viral vector used herein may be based on a varietyof paramyxoviruses including, without limitation, type I humanparainfluenza virus (HPI-V-1), type III human parainfluenza virus(HPIV-3), type III bovine parainfluenza virus (BPIV-3), Sendai virus(also referred to as “type I mouse parainfluenza virus”), or type xsimian parainfluenza virus (SPIV-10). These viruses may be naturallyoccurring, wild-type, mutant, laboratory-passaged, or artificiallyconstructed strains. Incomplete viruses such as, for example, the DIparticle (Willenbrink W. and Neubert W. J., J. Virol., 1994, 68,8413-8417) and synthesized oligonucleotides may also be utilized as amaterial for generating a paramyxovirus viral vector used herein. (Seee.g. US 2004/0005296).

Genes encoding proteins of a paramyxovirus include NP, P, M, F, HN, andL genes. The NP, P, M, F, HN, and L genes represent those encoding thenucleocapsid protein, phosphoprotein, matrix protein, fusion protein,hemagglutinin-neuraminidase, and large protein, respectively. The NPgene may also be indicated as the N gene. The aforementionedparamyxovirus proteins are well known in the art. For instance, theaccession numbers of each gene of the Sendai virus, for example,classified as a Respirovirus of Paramyxoviridae in the nucleotidesequence database, are M29343, M30202, M30203, M30204, M51331, M55565,M69046, and X17218 for NP gene; M30202, M30203, M30204, M55565, M69046,X00583, X17007, and X17008 for P gene; D11446, K02742, M30202, M30203,M30204, M69046, U31956, X00584, and X53056 for M gene; D00152, D11446,D17334, D17335, M30202, M30203, M30204, M69046, X00152, and X02131 for Fgene; D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808,and X56131 for HN gene; and D00053, M30202, M30203, M30204, M69040,X00587, and X58886 for L gene. (See e.g. US 2004/0005296). Note thatparamyxovirus-based vectors, such as Sendai virus-based vectors, usedherein may include modifications, such as deletions of endogenous viralproteins.

Paramyxovirus-based viral vectors are useful expression of a nucleicacid in a host cell. Since paramyxovirus vectors are not pathogenic inhumans, they can be suggested to be preferably utilized in clinicaltrials of human gene therapy in view of safety. It is a major obstaclein high efficient gene transfer that, in most cases, introduced DNA mustbe transported into the nucleus or nuclear membrane must be eliminatedfor the expression of an exogenous gene via plasmid DNA or such. In thecase of Sendai virus, however, expression of an exogenous gene is drivenby both cellular tubulin and its RNA polymerase (L protein) in thecytoplasm when viruses replicate. This suggests that the Sendai virusdoes not interact with chromosomes of host cells, which avoids riskssuch as cancerization and immortalization of cells. Furthermore, theSendai virus is known to be pathogenic in rodents causing pneumonia, butnot in humans, which is supported by studies showing that the intranasaladministration of the wild type Sendai virus does not do harm innonhuman primates (Hurwitz J. L. et al., Vaccine, 1997, 15, 533-540).These features suggest that Sendai virus vector can be utilized in humantherapy, and further, support the notion that Sendai virus vectors canbe a promising tool, in particular for use in contacting a human cellwith an agent that increases Zscan4 expression in a human cell. (Seee.g. US 2004/0005296). Accordingly, in certain embodiments, the virusvector is a Sendai virus vector. In some embodiments, the Sendai vectoris a temperature-sensitive Sendai vector. For example, the TS15temperature-sensitive Sendai vector is functional at 35° C., but can beinactivated when cultured at 37° C. (See e.g., Ban et al., Proc NatlAcad Sci USA. 2011; 108(34):14234-14239). Examples of furthertemperature-sensitive Sendai vectors include, without limitation, theTS7 and the TS13 Sendai vectors, which are functional at 32° C., 35° C.,and 37 C; but can be inactivated when cultured at 38° C. or 39° C. (Seee.g., Ban et al., Proc Natl Acad Sci USA. 2011; 108(34):14234-14239).Any other variant Sendai vector known in the art may also be used toexpress a Zscan4 of the present disclosure.

Further, retrovirus vectors (e.g., Moloney murine leukemia virus(MMLV)-based vectors) may also be used herein (See e.g. Takahashi etal., Cell 126:663-666, 2006; Takahashi et al., Cell 31:861-872, 2007;Okita et al., Nature 313-317, 2007; Park et al., Nature 451:141-146;U.S. Patent Application Publication No. 2009/0047263). Studies utilizinglentivirus-based vectors (Brambrink et al., Cell Stem Cell 2:151-159,2008; Wernig et al., Nat Biotechnol 26:916-924, 2008; Stadtfeld et al.,Science 322:945-949, 2008) demonstrated the advantage of these vectorsas being able to infect both dividing and non-dividing cells, therebyimproving the rate of cell transduction. In addition, lentiviruses canbe pseudotyped to expand viral tropism. For example, pseudotyping withvesicular stomatitis virus glycoprotein (VSVg) enables infection of awide range of cell types (Lai et al., J Assist Reprod Genet28(4):291-301, 2011). Lentiviruses also allow for both constitutive andinducible expression of the proteins. Examples of drug-induciblelentivirus expression systems are described by Hockmeyer et al. (CellStem Cell 3:346-353, 2008) and Wernig et al. (Nat Biotechnol 26:916-924,2008).

Lentiviruses include, but are not limited to, human immunodeficiencyvirus (such as HIV-1 and HIV-2), feline immunodeficiency virus, equineinfectious anemia virus and simian immunodeficiency virus. Otherretroviruses include, but are not limited to, human T-lymphotropicvirus, simian T-lymphotropic virus, murine leukemia virus, bovineleukemia virus and feline leukemia virus. Methods of generatingretrovirus and lentivirus vectors and their uses have been welldescribed in the art (see, for example, U.S. Pat. Nos. 7,211,247;6,979,568; 7,198,784; 6,783,977; and 4,980,289).

Non-integrating viral vectors, such as adenovirus vectors, have alsobeen used to deliver nucleic acid molecules encoding proteins to cells.For example adenovirus vectors, which remain in episomal form in cells,have been successfully used to deliver proteins into mouse fibroblastsand liver cells (Stadtfeld et al., Science 322:945-949, 2008).

In some embodiments, vectors containing the promoter and enhancerregions of the SV40 or long terminal repeat (LTR) of the Rous Sarcomavirus and polyadenylation and splicing signal from SV40 (Mulligan andBerg, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6; Gorman et al., 1982,Proc. Natl. Acad. Sci. USA 78:6777-81) are used. In some embodiments,the vector is a viral vector, such as an adenoviral vector, anadeno-associated virus (AAV), such as described in U.S. Pat. No.4,797,368 (Carter et al.) and in McLaughlin et al. (J. Virol.62:1963-73, 1988) and AAV type 4 (Chiorini et al. J. Virol. 71:6823-33,1997) and AAV type 5 (Chiorini et al. J. Virol. 73:1309-19, 1999), orretroviral vector (such as the Moloney Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus). Other viral transfection systems may also be utilized,including Vaccinia virus (Moss et al., 1987, Annu. Rev. Immunol.5:305-24), Bovine Papilloma virus (Rasmussen et al., 1987, MethodsEnzymol. 139:642-54) or members of the herpes virus group such asEpstein-Barr virus (Margolskee et al., 1988, Mol. Cell. Biol.8:2837-47). In addition, vectors may contain antibiotic selectablemarkers (such as neomycin, hygromycin or mycophenolic acid) to permitselection of transfected cells that exhibit stable, long-term expressionof the vectors (and therefore the Zscan4 nucleic acid).

The vectors can be maintained in the cells as episomal, freelyreplicating entities by using regulatory elements of viruses such aspapilloma (Sarver et al., 1981, Mol. Cell. Biol. 1:486) or Epstein-Barr(Sugden et al., 1985, Mol. Cell. Biol. 5:410). Alternatively, one canalso produce cell lines that have integrated the vector into genomicDNA. Both of these types of cell lines produce the gene product on acontinuous basis. One of skill in the art can also produce cell linesthat have amplified the number of copies of the vector (and therefore ofthe cDNA as well) to create cell lines that can produce high levels ofthe gene product.

Plasmid Vectors

In some instances, it is desirable to use non-viral vectors, such as toavoid integration into the host cell genome. Thus, Zscan4-encodingpolynucleotides can be delivered to a human cell using one or moreplasmid vectors. Plasmid vectors are episomally maintained and generallyexhibit a short duration of gene expression (Lai et al., J Assist ReprodGenet 28(4):291-301, 2011). As one example, Okita et al. (Science322:949-953, 2008) describe the use of the pCX plasmid, containing a CAGpromoter, for the expression of proteins in somatic cells.

Episomal plasmid vectors are a further option for introducingZscan4-encoding polynucleotides into a human cell. Episomal plasmidvectors are capable of replicating themselves autonomously asextrachromosomal elements, and therefore exhibit prolonged geneexpression in target cells. An episomal plasmid vector derived from theEpstein Barr virus (oriP/EBNA1) has been used to express proteins inhuman somatic cells (Yu et al., Science 324:797-801, 2009).

Selection of an appropriate vector is well within the capabilities ofone of skill in the art. Expression vectors typically contain an originof replication, a promoter, and optionally include specific genes toallow for phenotypic selection of the transformed cells (e.g. anantibiotic resistance cassette). Generally, the expression vector willinclude a promoter. The promoter can be inducible or constitutive. Thepromoter can also be tissue specific. Exemplary promoters include theCAG promoter, thymidine kinase promoter (TK), metallothionein I,polyhedron, neuron specific enolase, tyrosine hydroxylase, beta-actin,CMV immediate early promoter, or other promoters. Optionally, anenhancer element is also included, and can generally be located anywhereon the vector and still have an enhancing effect on gene expression.

Plasmid vectors can be introduced into human cells using any suitablemethod. In some embodiments, the vector is delivered to a cell bytransfection using a lipid of cationic polymer. In particular examples,the transfection reagent is LIPOFECTAMINE™, or a similar reagent. Inother examples, delivery is achieved using the nucleofectiontransfection technology (Amaxa, Cologne, Germany). This technology isbased on an electroporation technique using the NUCLEOFECTOR™ deliverydevice to introduce DNA directly into the host cell nucleus(Lakshmipathy et al., Stem Cells 22:531-543, 2004). In yet anotherexample, the transfection reagent includes poly-β-amino esters.

The transfer of DNA into human or other mammalian cells is aconventional technique. For example, an isolated Zscan4 nucleic acidsequence (for example as a naked DNA or as part of an expression vector)can be introduced into the recipient cells for example by precipitationwith calcium phosphate (Graham and vander Eb, 1973, Virology 52:466) orstrontium phosphate (Brash et al., 1987, Mol. Cell. Biol. 7:2013),electroporation (Neumann et al., 1982, EMBO J. 1:841), lipofection(Felgner et al., 1987, Proc. Natl. Acad. Sci. USA 84:7413), DEAE dextran(McCuthan et al., 1968, J. Natl. Cancer Inst. 41:351), microinjection(Mueller et al., 1978, Cell 15:579), protoplast fusion (Schafner, 1980,Proc. Natl. Acad. Sci. USA 77:2163-7), or pellet guns (Klein et al.,1987, Nature 327:70).

Excision Strategies

Excision of exogenous polynucleotides from genomic integration sites maybe desirable. Two excision-based methods have been previously described,CreloxP recombination and piggyBac transposition. Soldner et al. (Cell136:964-977, 2009) described the use of the Cre-lox system. Thisstrategy included positioning a loxP site in the 3′ LTR of a lentivirusvector that contained a Dox-inducible minimal CMV promoter to driveexpression of the reprogramming factors. During proviral replication,loxP was duplicated into the 5′ LTR, resulting in genomic integration ofthe reprogramming factors flanked by two loxP sites. Transientexpression of Cre-recombinase resulted in excision of the floxedreprogramming factors.

The piggyBac transposon is capable of excising itself without leavingany remnants of exogenous DNA in the cell genome (Elick et al., Genetica98:33-41, 1996; Fraser et al., Insect Mol Biol 5:141-151, 1996). Usingthis method, more than two proteins have been successfully produced inhuman cells by delivery of a polycistronic construct carrying geneslinked with a 2A peptide linker positioned between the piggyBactransposon 5′ and 3′ terminal repeats. Precise excision of theintegrated reprogramming genes is observed upon expression of thetransposase (Kaji et al., Nature 458:771-775, 2009; Wang et al., ProcNatl Acad Sci USA 105:9290-9295, 2008; Yusa et al., Nat Methods6:363-369, 2009).

mRNAs

Another strategy for introducing Zscan4 into human cells is by deliveryof synthetic mRNAs encoding Zscan4. It has been shown that a specificprotein can be efficiently produced by transfecting synthetic mRNAencoding the protein into cells (Warren et al., Cell Stem Cell7(5):618-630, 2010). In the study by Warren et al., the mRNA wasmodified to overcome innate antiviral responses. Transfection of mRNAswas carried out repeatedly—once a day for up to a few weeks tocompensate the transient nature of this method, because mRNAs werequickly degraded in the cells. This particular feature may beadvantageous for Zscan4, whose expression is required only for a shorttime (e.g., in the order of hours and days) to achieve the desiredeffects (i.e., extending telomeres and increasing genome stability). Incertain embodiments, synthetic mRNAs encoding Zscan4 are encapsulated ina viral envelope. Preferably, the viral envelope coating containsenvelope proteins that recognize cell surface receptors, and as suchincrease the efficiency of delivery of the synthetic Zscan4 mRNAs intothe cells. In certain embodiments, synthetic mRNAs encoding Zscan4 areencapsulated in nanoparticle or liposomes coated with viral envelopeproteins. Any suitable viral envelope known in the art may be used. Insome embodiments, the viral envelope is a Sendai virus envelope. Methodsof encapsulating polynucleotides, such as synthetic mRNAs, in viralenvelopes or viral envelope proteins are well known in the art.

Cells Including Zscan4 Polynucleotides

Further provided herein are isolated cells containing a Zscan4 nucleicacid molecule or Zscan4-containing vector as described herein. In someembodiments, the cell is a human cell. The origin of the human cell maybe from any suitable species. The human cell may include any type ofhuman cell described herein.

Zscan4 Polypeptides

In certain embodiments, an agent of the present disclosure thatincreases expression of Zscan4 is a Zscan4 polypeptide. A polypeptidemay refer to a polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The terms “polypeptide or“protein” are intended to encompass any amino acid sequence and includemodified sequences such as glycoproteins. The term “polypeptide” isspecifically intended to cover naturally occurring proteins, as well asthose which are recombinantly or synthetically produced.

Various Zscan4 polypeptides are known in the art and may be used in themethods described herein. One skilled in the art will appreciate thatthe various Zscan4 polypeptides described herein that retain Zscan4activity, such as the ability to increase telomere length and/or genomestability in a cell, may be used in the methods described herein.

Exemplary Zscan4 polypeptides are provided herein. For example, theamino acid sequence of the mouse Zscan4a polypeptide is set forth in SEQID NO: 11, the amino acid sequence of the mouse Zscan4b polypeptide isset forth in SEQ ID NO: 12, the amino acid sequence of the mouse Zscan4cpolypeptide is set forth in SEQ ID NO: 13, the amino acid sequence ofthe mouse Zscan4d polypeptide is set forth in SEQ ID NO: 14, the aminoacid sequence of the mouse Zscan4e polypeptide is set forth in SEQ IDNO: 15, and the amino acid sequence of the mouse Zscan4f polypeptide isset forth in SEQ ID NO: 16. Additionally, the amino acid sequence of thehuman ZSCAN4 polypeptide is set forth in SEQ ID NO: 17.

Zscan4 amino acid sequences from various other species are publicallyavailable, including dog Zscan4 (GenBank Accession Nos. XP.sub.—541370.2and XP.sub.—853650.1; SEQ ID NO: 18); cow Zscan4 (GenBank Accession No.XP.sub.—001789302.1; SEQ ID NO: 19); horse Zscan4 (GenBank Accession No.XP.sub.—001493994.1; SEQ ID NO: 20); gorilla Zscan4 (UniProt AccessionNo. A1YEQ9; SEQ ID NO: 31); bonobo Zscan4 (nucleotide sequence ofUniProt Accession No. A1YFX5; SEQ ID NO: 32); Bornean orangutan Zscan4(UniProt Accession No. A2T7G6; SEQ ID NO: 33), Sumatran orangutan Zscan4(UniProt Accession No. H2POE3; SEQ ID NO: 34); panda Zscan4 (UniProtAccession No. G1LE29; SEQ ID NO: 35); pig Zscan4 (UniProt Accession No.F1SCQ2; SEQ ID NO: 36); Northern white-cheeked gibbon Zscan4 (UniProtAccession No. G1RJD4; SEQ ID NO: 37); Rhesus macaque Zscan4 (UniProtAccession No. F7GH55; SEQ ID NO: 38); guinea pig Zscan4 (UniProtAccession No. HOV5E8; SEQ ID NO: 39); and Thirteen-lined ground squirrel(UniProt Accession No. I3N7T3; SEQ ID NO: 40). Each of the above-listedGenBank Accession numbers is herein incorporated by reference as itappears in the GenBank database on Aug. 11, 2009. Each of theabove-listed UniProt Accession numbers is herein incorporated byreference as it appears in the UniProt database on Mar. 15, 2013.

In some embodiments, the Zscan4 polypeptide is a human ZSCAN4polypeptide, or a homolog or ortholog thereof. In some embodiments, theZscan4 polypeptide is a mouse Zscan4 polypeptide or a homolog orortholog thereof. In some embodiments, the Zscan4 polypeptide is aZscan4a polypeptide, a Zscan4b polypeptide, a Zscan4c polypeptide, aZscan4d polypeptide, a Zscan4e polypeptide, or a Zscan4f polypeptide. Insome embodiments, the Zscan4 polypeptide is a dog Zscan4 polypeptide, ora homolog or ortholog thereof. In some embodiments, the Zscan4polypeptide is a cow Zscan4 polypeptide, or a homolog or orthologthereof. In some embodiments, the Zscan4 polypeptide is a horse Zscan4polypeptide, or a homolog or ortholog thereof. In some embodiments, theZscan4 polypeptide includes an amino acid sequence that is at least 70%,at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the amino acid sequencefrom any one of SEQ ID NOs: 11-20 and 31-40.

In some embodiments, the Zscan4 polypeptide is a human ZSCAN4polypeptide or homolog or ortholog thereof. In some embodiments, theZscan4 polypeptide includes an amino acid sequence that is at least 70%,at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the amino acid sequence ofSEQ ID NO: 17.

Fragments and variants of a Zscan4 polypeptide can readily be preparedby one of skill in the art using molecular techniques. A polypeptidefragment may refer to a portion of a polypeptide which exhibits at leastone useful epitope. The term “functional fragments of a polypeptide”refers to all fragments of a polypeptide that retain an activity of thepolypeptide, such as a Zscan4. Biologically functional fragments, forexample, can vary in size from a polypeptide fragment as small as anepitope capable of binding an antibody molecule to a large polypeptidecapable of participating in the characteristic induction or programmingof phenotypic changes within a cell, including affecting cellproliferation or differentiation. An epitope is a region of apolypeptide capable of binding an immunoglobulin generated in responseto contact with an antigen. Thus, smaller peptides containing thebiological activity of Zscan4, or conservative variants of Zscan4, arethus included as being of use. In some embodiments, a fragment of aZscan4 polypeptide includes at least 50, at least 100, at least 150, atleast 200, at least 250, at least 300, at least 350, at least 400, atleast 450 or at least 500 consecutive amino acids of the Zscan4polypeptide. In some embodiments, a fragment of Zscan4 is a fragmentthat confers a function of Zscan4 when transferred into a cell ofinterest, such as, but not limited to, increasing genome stabilityand/or increasing telomere length.

Minor modifications of the Zscan4 polypeptide primary amino acidsequences may result in peptides which have substantially equivalentactivity as compared to the unmodified counterpart polypeptide describedherein. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. All of the polypeptides produced bythese modifications are included herein.

One of skill in the art can readily produce fusion proteins including aZscan4 polypeptide and a second polypeptide of interest. Optionally, alinker can be included between the Zscan4 polypeptide and the secondpolypeptide of interest. Fusion proteins include, but are not limitedto, a polypeptide including a Zscan4 polypeptide and a marker protein.In some embodiments, the marker protein can be used to identify orpurify a Zscan4 polypeptide. Exemplary fusion proteins include, but arenot limited to, green fluorescent protein, six histidine residues, ormyc and a Zscan4 polypeptide.

One skilled in the art will appreciate that such variants, fragments,and fusions of Zscan4 useful for the disclosed methods are those thatretain Zscan4 activity (such as the ability to increase genome stabilityand increase telomere length or both in a human cell).

Various aspects of the present disclosure relate to substantiallypurified polypeptides. A substantially purified polypeptide may refer toa polypeptide which is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Polypeptides of the present disclosure, such as Zscan4 polypeptides, mayalso include conservative substitutions of the amino acids composing thepolypeptide. Conservative substitutions replace one amino acid withanother amino acid that is similar in size, hydrophobicity, etc.Examples of conservative substitutions are shown below:

Original Conservative Residue Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Variations in the cDNA sequence that result in amino acid changes,whether conservative or not, should be minimized in order to preservethe functional and immunologic identity of the encoded protein. Thus, inseveral non-limiting examples, a Zscan4 polypeptide includes at mosttwo, at most five, at most ten, at most twenty, or at most fiftyconservative substitutions. The immunologic identity of the protein maybe assessed by determining whether it is recognized by an antibody; avariant that is recognized by such an antibody is immunologicallyconserved. Any cDNA sequence variant will preferably introduce no morethan twenty, and preferably fewer than ten amino acid substitutions intothe encoded polypeptide.

In certain embodiments, Zscan4 polypeptides of the present disclosureare encapsulated in a viral envelope. Preferably, the viral envelopecoating contains envelope proteins that recognize cell surfacereceptors, and as such increase the efficiency of delivery of the Zscan4polypeptide into cells. In certain embodiments, Zscan4 polypeptides areencapsulated in nanoparticle or liposomes coated with viral envelopeproteins. Any suitable viral envelope known in the art may be used. Insome embodiments, the viral envelope is a Sendai virus envelope. Methodsof encapsulating polypeptides in viral envelopes or viral envelopeproteins are well known in the art.

Cells Including Zscan4 Polypeptides

Further provided herein are isolated cells containing a Zscan4polypeptide as described herein. In some embodiments, the cell is ahuman cell. The origin of the human cell may be from any suitablespecies. The human cell may include any type of human cell describedherein.

Compositions, Vectors and Cells Including a Modified Zscan4

Provided herein are isolated nucleic acid molecules encoding a modifiedZscan4 protein, where the protein has been modified so that the activityof the protein is regulatable (i.e., inducible or repressible). Forexample, the Zscan4 protein may be a fusion protein that contains aninducible receptor or ligand bind domain. Any inducible receptor and/orligand bind domain known in the art may be used. In some embodiments,the inducible receptor and/or ligand bind domain may include, withoutlimitation, an estrogen receptor (ER); a mutant estrogen receptor thatis sensitive to Tamoxifen or its metabolite 4-hydroxy-tamoxifen (4OHT),such as ERT or ERT2; a glucocorticoid receptor (GR) that isglucocorticoid receptor that is sensitive to mifepristone (MIFEPREX); adrug-regulatable ligand binding domain; and a steroid-induciblereceptor, such as an ecdysone-inducible receptor.

In certain embodiments, isolated nucleic acid molecules of the presentdisclosure encode a fusion protein, wherein the fusion protein includesa Zscan4 protein fused to an ERT2 protein. ERT2 is a mutated version ofthe ligand binding domain of human estrogen receptor. ERT2 does not bindits natural ligand (17β-estradiol) at physiological concentrations, butis highly sensitive to nanomolar concentrations of Tamoxifen or itsmetabolite 4-hydroxy-tamoxifen (4OHT) (Feil et al., Biochem Biophys ResCommun 237(3):752-757, 1997). A fusion protein may refer to a proteincontaining at least a portion of two different (heterologous) proteins.In some examples such proteins are generated by expression of a nucleicacid sequence engineered from nucleic acid sequences encoding at least aportion of two different (heterologous) proteins. To create a fusionprotein, the nucleic acid sequences must be in the same reading frameand contain no internal stop codons.

In some embodiments, the nucleic acid molecule encoding the Zscan4portion of a modified Zscan4 protein of the present disclosure, such asthe Zscan4-ERT2 fusion protein, is a human ZSCAN4, mouse Zscan4c, mouseZscan4d or mouse Zscan4f, or a functional fragment or variant thereof.The nucleic acid molecule encoding the modified Zscan4 protein, such asthe Zscan4-ERT2 fusion protein, may include any Zscan4 polynucleotide,or homolog, fragment, or variant thereof described herein. Functionalfragments and variants of Zscan4 include, for example, any Zscan4fragment or variant that retains one or more biological activities ofZscan4, such as the capacity to increase pluripotency of a stem cell,promote genomic stability or increase telomere length.

In some embodiments, the nucleic acid molecule encoding the Zscan4portion of a modified Zscan4 protein of the present disclosure, such asthe Zscan4-ERT2 fusion protein, may include, for example, the nucleotidesequence from any one of SEQ ID NOs: 1-10 and 21-30. In someembodiments, the nucleic acid molecule encoding the Zscan4 portion of amodified Zscan4 protein of the present disclosure, such as theZscan4-ERT2 fusion protein, is a mouse Zscan4 polynucleotide or ahomolog thereof. In some embodiments, the nucleic acid molecule encodingthe Zscan4 portion of a modified Zscan4 protein of the presentdisclosure, such as the Zscan4-ERT2 fusion protein, is a human ZSCAN4polynucleotide or a homolog thereof. In some embodiments, the nucleicacid molecule encoding the Zscan4 portion of a modified Zscan4 proteinof the present disclosure, such as the Zscan4-ERT2 fusion protein, is aZscan4a polynucleotide, a Zscan4b polynucleotide, a Zscan4cpolynucleotide, a Zscan4d polynucleotide, a Zscan4e polynucleotide, or aZscan4f polynucleotide. In some embodiments, the nucleic acid moleculeencoding the Zscan4 portion of a modified Zscan4 protein of the presentdisclosure, such as the Zscan4-ERT2 fusion protein, includes anucleotide sequence that is at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to a nucleotide sequence from any one of SEQ ID NOs: 1-10and 21-30.

In some embodiments, the nucleic acid molecule encoding the Zscan4portion of a modified Zscan4 protein of the present disclosure, such asthe Zscan4-ERT2 fusion protein, is a human ZSCAN4 polynucleotide orhomolog thereof. In some embodiments, the nucleic acid molecule encodingthe Zscan4 portion of a modified Zscan4 protein of the presentdisclosure, such as the Zscan4-ERT2 fusion protein, includes anucleotide sequence that is at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the nucleotide sequence of SEQ ID NO: 7.

In some embodiments, the nucleic acid molecule encoding a Zscan4-fusionprotein, such as the Zscan4-ERT2 fusion protein, includes a linkersequence between the Zscan4 and the inducible receptor and/or ligandbind domain sequence, such as the ERT2 coding sequence. Linkers are wellknown in the art and selection of an appropriate linker is well withinthe capabilities of one of ordinary skill in the art. A linker may referto one or more nucleotides or amino acids that serve as a spacer betweentwo molecules, such as between two nucleic acid molecules or twopeptides (such as in a fusion protein). In some embodiments, a linker is1 to 100 amino acids, such as 1 to 50 or 5 to 10 amino acids. In someembodiments, the linker is at least 2 amino acids (aa), at least 3, atleast 5, at least 10, at least 20, at least 50 or at least 100 aa, suchas 2 to 50 or 2 to 10 aa. In some embodiments, the linker includes theamino acid sequence Ala-Ser.

Also provided are vectors that include a modified Zscan4, such asZscan4-ERT2, encoding nucleic acid molecule disclosed herein. Anysuitable expression vector, such as an expression (plasmid) vector(e.g., pPyCAG-BstXI-IP), or viral vector (e.g., a paramyxovirus such asa Sendai virus, an adenovirus, adeno-associated virus, lentivirus orretrovirus vector), is contemplated. Numerous expression vectors andviral vectors are known in the art and the selection of an appropriatevector is well within the capabilities of one of ordinary skill in theart.

Further provided herein are isolated cells containing a modified Zscan4,such as a Zscan4-ERT2, nucleic acid molecule or vector as describedherein. In some embodiments, the cell is a human cell. The origin of thehuman cell may be from any suitable species. The human cell may includeany type of human cell described herein.

Compositions including a nucleic acid molecule or vector encoding amodified Zscan4 protein, such as a Zscan4-ERT2 fusion protein, are alsoprovided herein. The compositions may further include a carrier ordiluent, such as a pharmaceutically acceptable carrier or diluent.Modified Zscan4 proteins, such as Zscan4-ERT2 fusion proteins, encodedby the nucleic acid molecules and vectors described herein are furtherprovided.

Also provided herein are recombinant modified Zscan4 proteins, such asZscan4-ERT2 fusion proteins. In some embodiments, the recombinantmodified Zscan4 protein, such as the Zscan4-ERT2 fusion protein, ishuman ZSCAN4, mouse Zscan4c, mouse Zscan4d or mouse Zscan4f, or afunctional fragment or variant thereof. The Zscan4 portion of themodified Zscan4 protein, such as the Zscan4-ERT2 recombinant fusionprotein, may include any Zscan4 polypeptide, homolog, ortholog,fragment, or variant described herein. Functional fragments and variantsof Zscan4 include, for example, any Zscan4 fragment or variant thatretains one or more biological activities of Zscan4, such as thecapacity to increase genomic stability or increase telomere length.

In some embodiments, the Zscan4 protein portion of the modified Zscan4protein, such as the Zscan4-ERT2 fusion protein, is a mouse Zscan4polypeptide or a homolog or ortholog thereof. In some embodiments, theZscan4 protein portion of the modified Zscan4 protein, such asZscan4-ERT2 fusion protein, is a human ZSCAN4 polypeptide, or a homologor ortholog thereof. In some embodiments, the Zscan4 protein portion ofthe modified Zscan4 protein, such as Zscan4-ERT2 fusion protein, is aZscan4a polypeptide, a Zscan4b polypeptide, a Zscan4c polypeptide, aZscan4d polypeptide, a Zscan4e polypeptide, or a Zscan4f polypeptide. Insome embodiments, the Zscan4 protein portion of the modified Zscan4protein, such as Zscan4-ERT2 fusion protein, includes an amino acidsequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto an amino acid sequence from any one of SEQ ID Nos: 11-20 and 31-40.

In some embodiments, the Zscan4 protein portion of the modified Zscan4protein, such as Zscan4-ERT2 fusion protein, is a human ZSCAN4polypeptide or homolog or ortholog thereof. In some embodiments, theZscan4 protein portion of the modified Zscan4 protein, such asZscan4-ERT2 fusion protein, includes an amino acid sequence that is atleast 70%, at least 75%, at least 80%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to the aminoacid sequence of SEQ ID NO: 17.

Fragments and variants of a Zscan4 protein can readily be prepared byone of skill in the art using molecular techniques. In some embodiments,a fragment of a Zscan4 protein includes at least 50, at least 100, atleast 150, at least 200, at least 250, at least 300, at least 350, atleast 400, at least 450 or at least 500 consecutive amino acids of theZscan4 polypeptide. In a further embodiment, a fragment of Zscan4 is afragment that confers a function of Zscan4, such as, but not limited to,increasing genome stability and/or increasing telomere length.

Compositions including a modified Zscan4 protein, such as Zscan4-ERT2fusion protein, are also provided herein. The compositions may furtherinclude a carrier or diluent, such as a pharmaceutically acceptablecarrier or diluent, for example saline.

Compositions, Vectors and Cells Including Zscan4-ΔC

Also provided herein are isolated nucleic acid molecules encoding aZscan4 protein with a C-terminal truncation (referred to herein asZscan4-ΔC). The C-terminally truncated Zscan4 includes a deletion of atleast one zinc finger domain. Thus, in some embodiments, the Zscan4-ΔCprotein has a deletion of one, two, three or four zinc finger domains.

In some embodiments, the nucleic acid molecule encoding the Zscan4-ΔCprotein is a C-terminally truncated human ZSCAN4, mouse Zscan4c, mouseZscan4d or mouse Zscan4f. In some embodiments, the Zscan4-ΔC protein iseither human ZSCAN4 or mouse Zscan4c with a deletion of all four zincfinger domains. The nucleic acid molecule encoding the Zscan4-ΔC proteinmay contain a C-terminal truncation of any Zscan4 polynucleotidedescribed herein.

In some embodiments, the Zscan4 portion of the nucleic acid moleculeencoding the Zscan4-ΔC protein may include, for example, the nucleotidesequence from any one of SEQ ID Nos: 1-10 and 21-30. In someembodiments, the Zscan4 portion of the nucleic acid molecule encodingthe Zscan4-ΔC protein is a mouse Zscan4 polynucleotide or a homologthereof. In some embodiments, the Zscan4 portion of the nucleic acidmolecule encoding the Zscan4-ΔC protein is a human ZSCAN4 polynucleotideor a homolog thereof. In some embodiments, the Zscan4 portion of thenucleic acid molecule encoding the Zscan4-ΔC protein is a Zscan4apolynucleotide, a Zscan4b polynucleotide, a Zscan4c polynucleotide, aZscan4d polynucleotide, a Zscan4e polynucleotide, or a Zscan4fpolynucleotide. In some embodiments, the Zscan4 portion of the nucleicacid molecule encoding the Zscan4-ΔC protein includes a nucleotidesequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto a nucleotide sequence from any one of SEQ ID Nos: 1-10 and 21-30.

In some embodiments, the Zscan4 portion of the nucleic acid moleculeencoding the Zscan4-ΔC protein is a human ZSCAN4 polynucleotide orhomolog thereof. In some embodiments, the Zscan4 portion of the nucleicacid molecule encoding the Zscan4-ΔC protein includes a nucleotidesequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto the amino acid sequence of SEQ ID NO: 7.

The Zscan4-ΔC nucleic acid sequences contemplated herein includesequences that are degenerate as a result of the genetic code. There are20 natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included aslong as the amino acid sequence of the Zscan4-ΔC polypeptide encoded bythe nucleotide sequence is functionally unchanged.

Also provided are vectors that include a Zscan4-ΔC encoding nucleic acidmolecule disclosed herein. Any suitable expression vector, such as anexpression (plasmid) vector (e.g., pPyCAG-BstXI-IP), or viral vector(e.g., a paramyxovirus such as a Sendai virus, an adenovirus,adeno-associated virus, lentivirus or retrovirus vector), iscontemplated. Numerous expression vectors and viral vectors are known inthe art and the selection of an appropriate vector is well within thecapabilities of one of ordinary skill in the art.

Further provided herein are isolated cells containing a Zscan4-ΔCnucleic acid molecule or vector as described herein. In someembodiments, the cell is a human cell. The origin of the human cell maybe from any suitable species. The human cell may include any type ofhuman cell described herein.

Compositions including a nucleic acid molecule or vector encoding aZscan4ΔC protein are also provided herein. The compositions may furtherinclude a carrier or diluent, such as a pharmaceutically acceptablecarrier or diluent.

Zscan4-ΔC proteins encoded by the nucleic acid molecules and vectorsdescribed herein are further provided.

Also provided herein are recombinant Zscan4-ΔC proteins. In someembodiments, the recombinant Zscan4-ΔC protein is a C-terminallytruncated human ZSCAN4, mouse Zscan4c, mouse Zscan4d or mouse Zscan4f.The Zscan4 portion of a recombinant Zscan4-ΔC protein may include anyZscan4 polypeptide, homolog, ortholog, fragment, or variant describedherein.

In some embodiments, the Zscan4 protein portion of the recombinantZscan4-ΔC protein is a mouse Zscan4 polypeptide or a homolog or orthologthereof. In some embodiments, the Zscan4 protein portion of therecombinant Zscan4-ΔC protein is a human ZSCAN4 polypeptide, or ahomolog or ortholog thereof. In some embodiments, the Zscan4 proteinportion of the recombinant Zscan4-ΔC protein is a Zscan4a polypeptide, aZscan4b polypeptide, a Zscan4c polypeptide, a Zscan4d polypeptide, aZscan4e polypeptide, or a Zscan4f polypeptide. In some embodiments, theZscan4 protein portion of the recombinant Zscan4-ΔC protein includes anamino acid sequence that is at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the amino acid sequence from any one of SEQ ID Nos:11-20 and 31-40.

In some embodiments, the Zscan4 protein portion of the recombinantZscan4-ΔC protein is a human ZSCAN4 polypeptide or homolog or orthologthereof. In some embodiments, the Zscan4 protein portion of therecombinant Zscan4-ΔC protein includes an amino acid sequence that is atleast 70%, at least 75%, at least 80%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to the aminoacid sequence from SEQ ID NO: 17.

Further provided herein are isolated cells including a Zscan4-ΔC proteindisclosed herein. In some embodiments, the cells are human cells. Theorigin of the human cell may be from any suitable species. The humancell may include any type of human cell described herein.

Compositions including a Zscan4-ΔC protein are also provided herein. Thecompositions may further include a carrier or diluent, such as apharmaceutically acceptable carrier or diluent, for example saline.

Methods of Introducing Zscan4 Polypeptide into Human Cells

It is possible to introduce Zscan4 polypeptides by directly deliveringthe respective proteins to cells, such as human cells. Protein deliverycan be accomplished using, for example, electroporation, microinjection,cationic lipids or nanoparticles according to standard methods.Alternatively, the proteins can be modified by fusion with acell-penetrating peptide (CPP) to facilitate entry of the protein intothe cell. The use of CPPs and nanoparticles is discussed in greaterdetail herein.

Cell-Penetrating Peptides (CPPs)

CPPs are a family of polypeptides that facilitate transduction ofproteins, nucleic acids or other compounds across membranes in areceptor-independent manner (Wadia and Dowdy, Curr. Protein Pept. Sci.4(2):97-104, 2003). Typically, CPPs are short polycationic sequencesthat can facilitate cellular uptake of compounds to which they arelinked into endosomes of cells. Examples of CPPs include poly-argininetags and protein transduction domains. Any protein transduction domainknown in the art may be used. Examples of suitable protein transductiondomains include, without limitation, HIV Tat, HIV Vpr, HIV Vp22,homeodomains (HD) from HD-containing proteins, and synthetic proteintransduction domains.

The capacity of certain peptides to deliver proteins or nucleic acidsinto cells was originally described for the HIV-encoded Tat protein,which was shown to cross membranes and initiate transcription. It wasthen discovered that the portion of the Tat protein that was requiredfor the transduction of the protein was only an 11 amino acidpolypeptide, referred to as the Tat peptide. When fused with otherproteins, the Tat peptide has been demonstrated to deliver theseproteins, varying in size from 15 to 120 kDa, into cells in tissueculture (Frankel and Pabo, Cell 55(6):1189-93, 1988; Green andLoewenstein, J. Gen. Microbiol. 134(3):849-55, 1988; Vives et al., J.Biol. Chem. 272(25):16010-7, 1997; Yoon et al., J. Microbiol.42(4):328-35, 2004; Cal et al., Eur. J. Pharm. Sci. 27(4):311-9, 2006).

Other known CPPs include PENETRATIN™, a 16 amino acid peptide derivedfrom the third helix of the Drosophila Antennapedia homeobox gene (U.S.Pat. No. 5,888,762; Derossi et al., J. Biol. Chem. 269:10444-10450,1994; Schwarze et al., Trends Pharmacol. Sci. 2/:45-48, 2000);transportan, a 27 amino acid chimeric peptide comprised of 12 aminoacids from the N-terminus of the neuropeptide galanin and the 14-aminoacid protein mastoparan, connected via a lysine (U.S. Pat. No.6,821,948; Pooga, FASEB J. 12:67-77, 1998; Hawiger, Curr. Opin. Chem.Biol. 3:89-94, 1999); peptides from the VP22 protein of herpes simplexvirus (HSV) type 1 (Elliott et al., Cell 88:223-233, 1997); the UL-56protein of HSV-2 (U.S. Pre-Grant Publication No. 2006/0099677); and theVpr protein of HIV-1 (U.S. Pre-Grant Publication No. 2005/0287648). Inaddition, a number of artificial peptides also are known to function asCPPs, such as poly-arginine, poly-lysine and others (see, for example,U.S. Pre-Grant Publication Nos. 2006/0106197; 2006/0024331;2005/0287648; and 2003/0125242; Zhibao et al., Mol. Ther. 2:339-347,2000; and Laus et al. Nature Biotechnol. 18:1269-1272, 2000).

Zhou et al. (Cell Stem Cell 4:381-384, 2009) describe the successful useof poly-arginine peptide tags. In addition, Kim et al. (Cell Stem Cell4:472-476, 2009) describe the successful use of the HIV-TAT proteintransduction domain to deliver proteins to human fetal fibroblasts.

Nanoparticles

Nanoparticles are submicron (less than about 1000 nm) sized drugdelivery vehicles that can carry encapsulated drugs such as syntheticsmall molecules, proteins, peptides, cells and nucleic acid basedbiotherapeutics for either rapid or controlled release. A variety ofmolecules (e.g., proteins, peptides and nucleic acid molecules) can beefficiently encapsulated in nanoparticles using processes well known inthe art. A molecule encapsulated in a nanoparticle may refer to amolecule (such as a Zscan4 nucleic acid or protein) that is eithercontained within the nanoparticle or attached to the surface of thenanoparticle, or a combination thereof.

In some examples, an agent that increases Zscan4 expression in a humancell is encapsulated by a nanoparticle to aid in delivery to the cells.Suitable nanoparticles for use with the disclosed methods are known inthe art and are described briefly below.

The nanoparticles for use with the methods described herein can be anytype of biocompatible nanoparticle, such as biodegradable nanoparticles,such as polymeric nanoparticles, including, but not limited topolyamide, polycarbonate, polyalkene, polyvinyl ethers, and celluloseether nanoparticles. In some embodiments, the nanoparticles are made ofbiocompatible and biodegradable materials. In some embodiments, thenanoparticles include, but are not limited to nanoparticles includingpoly(lactic acid) or poly(glycolic acid), or both poly(lactic acid) andpoly(glycolic acid). In some embodiments, the nanoparticles arepoly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles.

Other biodegradable polymeric materials are contemplated for use withthe methods described herein, such as poly(lactic acid) (PLA) andpolyglycolide (PGA). Additional useful nanoparticles includebiodegradable poly(alkylcyanoacrylate) nanoparticles (Vauthier et al.,Adv. Drug Del. Rev. 55: 519-48, 2003).

Various types of biodegradable and biocompatible nanoparticles, methodsof making such nanoparticles, including PLGA nanoparticles, and methodsof encapsulating a variety of synthetic compounds, proteins and nucleicacids, has been well described in the art (see, for example, U.S.Publication No. 2007/0148074; U.S. Publication No. 20070092575; U.S.Patent Publication No. 2006/0246139; U.S. Pat. Nos. 5,753,234;7,081,489; and PCT Publication No. WO/2006/052285).

Retinoids

In certain embodiments, an agent of the present disclosure thatincreases expression of Zscan4 is a retinoid. A retinoid may refer to aclass of chemical compounds that are related chemically to vitamin A.Retinoids are used in medicine, primarily due to the way they regulateepithelial cell growth. Retinoids have many important and diversefunctions throughout the body including roles in vision, regulation ofcell proliferation and differentiation, growth of bone tissue, immunefunction, and activation of tumor suppressor genes. Examples ofretinoids include, but are not limited to, all-trans retinoic acid(atRA), 9-cis retinoic acid (9-cis RA), 13-cis RA and vitamin A(retinol).

Various retinoids are known in the art and may be used in the methodsdescribed herein. Retinoids may include, without limitation, all-transretinoic acid, 9-cis retinoic acid, 13-cis retinoic acid, or vitamin A.

Agents that Induce Oxidative Stress

In certain embodiments, an agent of the present disclosure thatincreases expression of Zscan4 is an agent that induces oxidativestress. Oxidative stress may refer to an imbalance between theproduction of reactive oxygen species and a biological system's abilityto readily detoxify the reactive intermediates or to repair theresulting damage. Disturbances in the normal redox state of tissues cancause toxic effects through the production of peroxides and freeradicals that damage all components of the cell, including proteins,lipids, and DNA. In some embodiments of the disclosed methods, the agentthat induces oxidative stress is hydrogen peroxide (H₂O₂).

Various agents that induce oxidative stress are known in the art and maybe used in the methods described herein.

Increasing Telomere Length and Genome Stability

Certain aspects of the present disclosure relate to methods ofincreasing telomere length and/or increasing genome stability in one ormore human cells by, for example, contacting one or more human cellswith an agent that increases expression (e.g., protein expression) ofZscan4 in the one or more human cells. As disclosed herein, a transientincrease in expression and/or increased expression for only a shortperiod of time (e.g., from about 1 hour to about 23 hours, or from about1 day to about 10 days) is sufficient to increase telomere length and/orincrease genome stability in the human cells. Moreover, in someembodiments, repeating the transient increase in Zscan4 expression inthe human cells enhances the effects of the increase in Zscan4expression. In certain embodiments, the transient increase in Zscan4expression is repeated every 4 hours, every 8 hours, every 12 hours,every 16 hours, every 24 hours, every 32 hours, every 40 hours, every 48hours, every three days, every four days, every five days, every sixdays, every week, every two weeks, every three weeks, every four weeks,every month, every two months, every three months, every four months,every six months, every seven months, every eight months, every ninemonths, every 10 months, every 11 months, every year, every two years,every three years, every four years, every five years, every six years,every seven years, every eight years, every nine years, every 10 years,every 11 years, every 12 years, every 13 years, every 14 years, every 15years, every 16 years, every 17 years, every 18 years, every 19 years,every 20 years, every 21 years, every 22 years, every 23 years, every 24years, every 25 years, every 26 years, every 27 years, every 28 years,every 29 years, every 30 years, every 35 years, every 40 years, every 45years, or every 50 years.

Contacting may refer to placement in direct physical association;including both in solid and liquid form. “Contacting” may be usedinterchangeably with “exposed.” In some cases, “contacting” includestransfecting, such as transfecting a nucleic acid molecule into a cell.Methods of measuring genome stability and telomere length are routine inthe art, and the disclosure is not limited to particular methods. Theparticular examples provided herein are exemplary.

In some embodiments, telomere length is increased in human cellscontacted with an agent that increases Zscan4 protein expression in thehuman cells, by at least 1.5 fold, at least 1.6 fold, at least 1.7 fold,at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, atleast 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, atleast 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold,at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0fold, at least 9.5 fold, at least 10 fold, at least 100 fold, at least200 fold, at least 300 fold, at least 400 fold, at least 500 fold, atleast 600 fold, at least 700 fold, at least 800 fold, at least 900 fold,at least 1,000 fold, at least 2,000 fold, at least 3,000 fold, at least4,000 fold, at least 5,000 fold, at least 6,000 fold, at least 7,000fold, at least 8,000 fold, at least 9,000 fold, at least 10,000 fold, atleast 25,000 fold, at least 50,000 fold, at least 75,000 fold, at least100,000 fold, at least 125,000 fold, at least 150,000 fold, at least175,000 fold, at least 200,000 fold, at least 225,000 fold, at least250,000 fold, at least 275,000 fold, at least 300,000 fold, at least325,000 fold, at least 350,000 fold, at least 375,000 fold, at least400,000 fold, at least 425,000 fold, at least 450,000 fold, at least475,000 fold, at least 500,000 fold, at least 525,000 fold, at least550,000 fold, at least 575,000 fold, at least 600,000 fold, at least625,000 fold, at least 650,000 fold, at least 675,000 fold, at least700,000 fold, at least 725,000 fold, at least 750,000 fold, at least775,000 fold, at least 800,000 fold, at least 825,000 fold, at least850,000 fold, at least 875,000 fold, at least 900,000 fold, at least925,000 fold, at least 950,000 fold, at least 975,000 fold, or at least1,000,000 fold, for example, relative to Zscan4 protein expression in acorresponding human cell that has not contacted with the agent thatincreases Zscan4 expression. Any method known in the art and disclosedherein for determining Zscan4 protein expression in a cell, or forquantifying the number of proteins (i.e., protein stoichiometry) percell may be used.

In other embodiments, methods of increasing telomere length and/orgenome stability in one or more human cells includes contacting the oneor more human cells with a nucleic acid molecule or vector encoding amodified Zscan4 protein of the present disclosure, such as a Zscan4-ERT2fusion protein. In other embodiments, the method includes contacting thehuman cell or human cell population with a modified Zscan4 protein ofthe present disclosure, such as a Zscan4-ERT2 fusion protein.

In yet other embodiments, methods increasing telomere length and/orgenome stability in one or more human cells includes contacting thehuman cell or human cell population with a nucleic acid molecule orvector encoding a Zscan4-ΔC protein disclosed herein. In otherembodiments, the method includes contacting the human cell or human cellpopulation with a Zscan4-ΔC protein disclosed herein.

Methods of delivering nucleic acid molecules encoding Zscan4-ERT2 orZscan4-ΔC, and Zscan4-ERT2 or Zscan4-ΔC proteins to human cells areknown in the art and are described herein.

In some embodiments, telomere length is increased in human cells by atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 110%, atleast 115%, at least 120%, at least 125%, at least 130%, at least 135%,at least 140%, at least 145%, at least 150%, at least 155%, or at least160%, for example, relative to a human cell that has not been contactedwith a modified Zscan4 protein, such as a Zscan4-ERT2 or Zscan4-ΔCprotein, or a nucleic acid encoding a modified Zscan4 protein, such as aZscan4-ERT2 or Zscan4-ΔC protein (or compared to a value or range ofvalues expected in a human cell that has not undergone frequentactivation of Zscan4). In some embodiments, telomere length is increasedin human cells by at least at least 1.2-fold, at least 1.3-fold, atleast 1.4-fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold,at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1fold, at least 2.15 fold, at least 2.2 fold, at least 2.25 fold, atleast 2.3 fold, at least 2.35 fold, at least 2.4 fold, at least 2.45fold, at least 2.5 fold, at least 2.55 fold, at least 3.0 fold, at least3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, atleast 5.5 fold, at least 6.0 fold, at least 6.5 fold, at least 7.0 fold,at least 7.5 fold, at least 8.0 fold, at least 8.5 fold, at least 9.0fold, at least 9.5 fold, or at least 10 fold, for example, relative to ahuman cell that has not been contacted with a modified Zscan4 protein,such as a Zscan4-ERT2 or Zscan4-AC protein, or a nucleic acid encoding amodified Zscan4 protein, such as a Zscan4-ERT2 or Zscan4-ΔC protein (orcompared to a value or range of values expected in a human cell that hasnot undergone frequent activation of Zscan4).

In some embodiments, telomere length is measured in the one or morehuman cells that have been contacted with an agent that increases Zscan4expression in the human cells. In some examples, telomere length isincreased in a human cell if the length of the telomeres is greater, forexample, relative to telomere length in a human cell not contacted withthe agent that increases Zscan4 expression. For example, telomere lengthcan be detected in a human cell by fluorescence in situ hybridization(FISH), quantitative FISH (Q-FISH), or telomere qPCR.

Genome Stability

In some embodiments, genome stability is increased in one or more humancells contacted with an agent of the present disclosure that increasesexpression of Zscan4 in the human cells by at least 20%, at least 40%,at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, atleast 95%, or at least 98%, for example, relative to a correspondinghuman cell not contacted with the agent that increases expression ofZscan4.

Methods of measuring genome stability are routine in the art, and thedisclosure is not limited to particular methods. The particular examplesprovided herein are exemplary.

In some examples, genome stability in a human cell, such as a human cellcontacted with an agent that increases Zscan4 expression (e.g., agentthat increases expression of Zscan4, such as a Zscan4 nucleic acid,Zscan4 protein, Zscan4-ERT or Zscan4-ΔC), is measured by performingkaryotype analysis. Genome stability is increased if the presence ofkaryotype abnormalities (such as chromosome fusions and fragmentations)is decreased or even absent, for example relative to a cell that has notbeen contacting with the agent that increases expression of Zscan4. Forexample, karyotype analysis can be performed in human cells by inducingmetaphase arrests, then preparing metaphase chromosome spreads.

In some examples, genome stability in a human cell, such as a human cellcontacted with a Zscan4, Zscan4-ERT, or Zscan4-ΔC protein or nucleicacid, is measured by measuring sister chromatid exchange (SCE). Genomestability is increased if the presence of SCE is decreased relative to acontrol, such as a stem cell that has not undergone frequent activationof Zscan4. For example, SCE can be measured in a stem cell by detectingSCE in a metaphase spread.

Therapeutic Uses of Zscan4

It is disclosed herein that expression of Zscan4 increases telomerelength, increases genome stability, corrects genome and/or chromosomeabnormalities, protects cells against DNA damage, and/or enhances DNArepair. DNA repair may refer to a collection of processes by which acell identifies and corrects damage to the DNA molecules that encode itsgenome. Thus, provided herein are methods related to increasing theexpression of Zscan4 in human cells to increase genome stability,protect cells against DNA damage, enhance DNA repair, and increasetelomere length in human cells.

Methods are provided for treating subjects in need of human celltherapy, such as a subject having human cells in need of telomerelengthening or of correcting telomere and/or chromosome abnormalities.These methods may include the use of human cells. In some embodiments,the methods may include contacting one or more human cells with an agentthat increases expression of Zscan4 in the human cell. Increasedexpression of Zscan4 in the one or more human cells induces telomerelengthening in the one or more human cells as compared to one or morecorresponding human cells that are not contacted with the agent.

Methods are provided for treating a subject in need of telomerelengthening. In some embodiments, the methods may include contacting oneor more human cells in the subject with an agent that increasesexpression of Zscan4 in the one or more human cells, where increasedexpression of Zscan4 induces telomere lengthening in the one or morehuman cells. In some embodiments, the methods may include isolating oneor more human cells, contacting the one or more human cells with anagent that increases expression of Zscan4 in the one or more humancells, and administering the contacted one or more human cells to thesubject. Increasing expression of Zscan4 induces telomere lengthening inthe one or more human cells.

Methods are also provided for treating a disease or condition associatedwith a telomere and/or chromosome abnormality. In some embodiments, themethods may include administering to a subject in need thereof an agentthat increases expression of Zscan4 in one or more human cells in thesubject, where increasing expression of Zscan4 induces telomerelengthening and/or correction of telomere and/or chromosomeabnormalities in the one or more human cells to treat to treat the totreat the disease or condition associated with a telomere and/orchromosome abnormality. In some embodiments, the methods may includeisolating one or more human cells from a subject suffering from adisease or condition associated with a telomere and/or chromosomeabnormality, contacting the one or more human cells with an agent thatincreases expression of Zscan4 in the one or more human cells, andadministering the contacted one or more human cells to the subject totreat the associated with a telomere and/or chromosome abnormality.Increasing expression of Zscan4 induces telomere lengthening and/orcorrection of telomere and/or chromosome abnormalities in the one ormore human cells. In some embodiments, the methods may include isolatinghuman bone marrow cells from a subject suffering from a disease orcondition associated with a telomere and/or chromosome abnormality,contacting the human bone marrow cells with an agent that increasesexpression of Zscan4 in the human bone marrow cells, and engrafting thecontacted human bone marrow cells into the subject to treat the diseaseor condition associated with a telomere and/or chromosome abnormality.Increasing expression of Zscan4 induces telomere lengthening and/orcorrection of telomere and/or chromosome abnormalities in the human bonemarrow cells.

Methods are also provided for increasing genome stability of one or morehuman cells. In some embodiments, the methods may include contacting theone or more human cells with an agent that increases expression ofZscan4 in the one or more human cells, where increased expression ofZscan4 increases genome stability in the one or more human cells ascompared to one or more corresponding human cells that are not contactedwith the agent.

Methods are also provided for increasing DNA repair capacity of one ormore human cells. In some embodiments, the methods may includecontacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, where increasedexpression of Zscan4 increases DNA repair capacity in the one or morehuman cells as compared to one or more corresponding human cells thatare not contacted with the agent.

Methods are also provided for rejuvenating one or more human cellsand/or extending lifespan of one or more human cells. In someembodiments, the methods may include contacting the one or more humancells with an agent that increases expression of Zscan4 in the one ormore human cells, where increased expression of Zscan4 rejuvenatesand/or extends the lifespan of the one or more human cells as comparedto one or more corresponding human cells that are not contacted with theagent.

Methods are provided for rejuvenating a tissue or organ in a subjectand/or extending lifespan of a tissue or organ in a subject. In someembodiments, the methods may include administering to a subject in needthereof an agent that increases expression of Zscan4 in the tissue ororgan, wherein increasing expression of Zscan4 rejuvenates and/orextends the lifespan of the tissue or organ.

Methods are provided for treating a disease or condition associated withone or more deficiencies in resident tissue stem cells (i.e., tissuestem cells resident in the organ and/or tissue of the human body), suchas Duchenne muscular dystrophy. In some embodiments, the methods mayinclude administering to resident tissue stem cells in a subject in needthereof an agent that increases expression of Zscan4 in the residenttissue stem cells, wherein increasing expression of Zscan4 prevents thedeterioration of the cells.

Methods are provided for rejuvenating a subject in need thereof and/orextending lifespan of a subject in need thereof. In some embodiments,the methods may include administering to the subject an agent thatincreases expression of Zscan4, wherein increasing expression of Zscan4rejuvenates and/or extends the lifespan of the subject. In certainembodiments, the methods may include isolating one or more human cellsfrom the subject; contacting the one or more human cells with an agentthat increases expression of Zscan4 in the one or more human cells,where increasing expression of Zscan4 rejuvenates and/or extends thelifespan of the one or more human cells, and administering the contactedone or more human cells to the subject to rejuvenate and/or extend thelifespan of the subject. In some embodiments, administering the agentthat increases expression of Zscan4 may include, without limitation,injecting the agent to each organ and tissue of the body, injecting theagent to the circulating blood of the subject, injecting the agent intothe cerebrospinal fluids of the subject, injecting the agent into thelymphatic system of the subject, administering the agent into the lungtissue of the subject, administering the agent into the digestive organsand tissues, including the esophagus, stomach, and intestines of thesubject, injecting the agent into portal veins of the subject, andtopically administering the agent the skin and skin appendages, such ashair follicles and sweat glands to rejuvenate the tissue stem cells,progenitor cells, and/or terminally differentiated cells residing in thetreated organ and/or tissue. It is believed that the overall effects ofthe rejuvenation of tissue stem cells, progenitor cells, and/orterminally differentiated cells in the treated subject are therejuvenation of the subject and/or the slowing down of the aging processof the subject. It is also believed that rejuvenation of tissue stemcells, progenitor cells, and/or terminally differentiated cells in thetreated subject will result in lifespan extension of the subject.

For example, provided herein is a method of treating a subject withcancer by administering to the subject a Zscan4 polypeptide orpolynucleotide. A subject may refer to living multi-cellular vertebrateorganisms, a category that includes human and non-human mammals. In someembodiments, the method further includes selecting a patient in need ofsuch therapy, such as a subject that has been diagnosed with cancer.Cancer may refer to a malignant tumor characterized by abnormal oruncontrolled cell growth. Other features often associated with cancerinclude metastasis, interference with the normal functioning ofneighboring cells, release of cytokines or other secretory products atabnormal levels and suppression or aggravation of inflammatory orimmunological response, invasion of surrounding or distant tissues ororgans, such as lymph nodes, etc. Metastatic disease may refer to cancercells that have left the original tumor site and migrate to other partsof the body for example via the bloodstream or lymph system.

In some embodiments of the methods disclosed herein, the subject isadministered a Zscan4 polynucleotide. In some examples, the subject isadministered a vector including a Zscan4 polynucleotide. Methods ofgenerating and using Zscan4-expresssing vectors are described in othersections of the application. In some embodiments, the Zscan4polynucleotide (or vector including the Zscan4 polynucleotide) isadministered directly to tumor cells to tumor tissue, such as byinjection.

In some embodiments, the subject is administered a Zscan4 polypeptide.In some embodiments, a Zscan4 polynucleotide and/or Zscan4 polypeptideof the present disclosure is encapsulated by a nanoparticle to aid indelivery of the Zscan4 polynucleotide, Zscan4 polypeptide, and/or agentthat induces Zscan4 expression to tumor cells. Suitable nanoparticlesfor use with the disclosed methods are known in the art and aredescribed below.

Nanoparticles are submicron (less than about 1000 nm) sized drugdelivery vehicles that can carry encapsulated drugs such as syntheticsmall molecules, proteins, peptides and nucleic acid basedbiotherapeutics for either rapid or controlled release. A variety ofmolecules (e.g., proteins, peptides and nucleic acid molecules) can beefficiently encapsulated in nanoparticles using processes well known inthe art.

The nanoparticles for use with the compositions and methods describedherein can be any type of biocompatible nanoparticle, such asbiodegradable nanoparticles, such as polymeric nanoparticles, including,but not limited to polyamide, polycarbonate, polyalkene, polyvinylethers, and cellulose ether nanoparticles. In some embodiments, thenanoparticles are made of biocompatible and biodegradable materials. Insome embodiments, the nanoparticles include, but are not limited tonanoparticles including poly(lactic acid) or poly(glycolic acid), orboth poly(lactic acid) and poly(glycolic acid). In some embodiments, thenanoparticles are poly(D,L-lactic-co-glycolic acid) (PLGA)nanoparticles.

PLGA is an FDA-approved biomaterial that has been used in resorbablesutures and biodegradable implants. PLGA nanoparticles have also beenused in drug delivery systems for a variety of drugs via numerous routesof administration including, but not limited to, subcutaneous,intravenous, ocular, oral and intramuscular. Administration may refer toproviding or giving a subject an agent by any effective route. Anexemplary route of administration includes, but is not limited to,injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, intravenous or intra-arterial). PLGA degrades into itsmonomer constituents, lactic and glycolic acid, which are naturalbyproducts of metabolism, making the material highly biocompatible. Inaddition, PLGA is commercially available as a clinical-grade materialfor synthesis of nanoparticles.

Other biodegradable polymeric materials are contemplated for use withthe compositions and methods described herein, such as poly(lactic acid)(PLA) and polyglycolide (PGA). Additional useful nanoparticles includebiodegradable poly(alkylcyanoacrylate) nanoparticles (Vauthier et al.,Adv. Drug Del. Rev. 55: 519-48, 2003). Oral adsorption also may beenhanced using poly(lactide-glycolide) nanoparticles coated withchitosan, which is a mucoadhesive cationic polymer. The manufacture ofsuch nanoparticles is described, for example, by Takeuchi et al. (Adv.Drug Del. Rev. 47: 39-54, 2001).

Among the biodegradable polymers currently being used for humanapplications, PLA, PGA, and PLGA are known to be generally safe becausethey undergo in vivo hydrolysis to harmless lactic acid and glycolicacid. These polymers have been used in making sutures when post-surgicalremoval is not required, and in formulating encapsulated leuprolideacetate, which has been approved by the FDA for human use (Langer andMose, Science 249:1527, 1990); Gilding and Reed, Polymer 20:1459, 1979;Morris, et al., Vaccine 12:5, 1994). The degradation rates of thesepolymers vary with the glycolide/lactide ratio and molecular weightthereof. Therefore, the release of the encapsulated molecule (such as aprotein or peptide) can be sustained over several months by adjustingthe molecular weight and glycolide/lactide ratio of the polymer, as wellas the particle size and coating thickness of the capsule formulation(Holland, et al., J. Control. Rel. 4:155, 1986).

In some embodiments, the nanoparticles for use with the compositions andmethods described herein range in size from about 50 nm to about 1000 nmin diameter. In some embodiments, the nanoparticles are less than about600 nm. In some embodiments, the nanoparticles are about 100 to about600 nm in diameter. In some embodiments, the nanoparticles are about 200to about 500 nm in diameter. In some embodiments, the nanoparticles areabout 300 to about 450 nm in diameter. One skilled in the art wouldreadily recognize that the size of the nanoparticle may vary dependingupon the method of preparation, clinical application, and imagingsubstance used.

Various types of biodegradable and biocompatible nanoparticles, methodsof making such nanoparticles, including PLGA nanoparticles, and methodsof encapsulating a variety of synthetic compounds, proteins and nucleicacids, has been well described in the art (see, for example, U.S.Publication No. 2007/0148074; U.S. Publication No. 20070092575; U.S.Patent Publication No. 2006/0246139; U.S. Pat. Nos. 5,753,234;7,081,489; and PCT Publication No. WO/2006/052285).

In some embodiments, one or more human cells are contacted with an agentthat increases expression of Zscan4 in the one or more human cells. Asused herein, a human cell that has been contacted with an agent thatincreases the expression of Zscan4 is referred to as a “Zscan4+ humancell”. As disclosed herein, “Zscan4+ cells” include, without limitation,cells that transiently express Zscan4. That is, Zscan4+ cells do notnecessarily continue to have a contact with Zscan4 or continuallyexpress Zscan4. As disclosed in some embodiments of the presentdisclosure, the action of Zscan4 is rapid and usually requires onlytransient and short contact (e.g., in the order of hours to days). Inthe case of telomeres, once telomeres are extended by the transientZscan4 action, Zscan4 is not required for a long time as the telomeresget shorter only gradually. Accordingly, “Zscan4+ human cells” caninclude both cells that are contacted with an agent of the presentdisclosure that increases expression of Zscan4, and cells that werecontacted with an agent of the present disclosure that increasesexpression of Zscan4, but are no longer in contact with the agent.Zscan4+ human cells may be administered to a subject in need thereof totreat a disorder or disease.

Subjects that can be treated using the methods provided herein mayinclude mammalian subjects, such as a veterinary or human subject.Subjects may include a fetus, newborns, infants, children, and/oradults. In some embodiments, the subject to be treated is selected, suchas selecting a subject that would benefit from human cell therapy,particularly therapy that includes administration of Zscan4+ human cellsand/or an agent that increases Zscan4 expression in human cells.

Examples of disorders or diseases that can benefit from administrationof Zscan4+ human cells and/or an agent that increases Zscan4 expressionin human cells include those disorders or diseases that are associatedwith telomere-shortening. Further examples of disorders or diseases thatcan benefit from administration of Zscan4+ human cells and/or an agentthat increases Zscan4 expression in human cells include cancer,autoimmune diseases, and diseases in which cell regeneration isbeneficial, such as neurologic injuries or a neurodegenerativedisorders, as well as blindness, deafness, tooth loss, arthritis,myocardial infarctions, bone marrow transplants, baldness, Crohn'sdisease, diabetes, muscular dystrophy, and Duchenne muscular dystrophy.In particular examples, a subject having one or more of these disordersis selected for the treatments herein disclosed.

In some embodiments, a subject of the present disclosure in need ofZscan4 treatment has a disease or condition associated with a telomereabnormality. A telomere abnormality may refer to any change in atelomere, such as telomere shortening, disruption of telomeric DNArepeats, or telomere DNA mutation, that disrupts one or more telomerefunction. Exemplary diseases or conditions associated with telomereabnormality that may benefit from Zscan4+ human cells and/or an agentthat increases Zscan4 expression in human cells may include, withoutlimitation, diseases of telomere shortening, bone marrow failuresyndromes, age-related telomere shortening diseases, and premature agingdisorders.

A disease or condition of telomere shortening that may benefit fromZscan4+ human cells and/or an agent that increases Zscan4 expression inhuman cells may include, without limitation, dyskeratosis congenita,Hoyeraal-Hreidarsson syndrome, Revesz syndrome, Coats plus syndrome,idiopathic pulmonary fibrosis, liver cirrhosis, pancreatic fibrosis, anddegenerative diseases, such as Alzheimer's disease and osteoarthritis.

A disease or condition of a bone marrow failure syndrome that maybenefit from Zscan4+ human cells and/or an agent that increases Zscan4expression in human cells may include, without limitation, Fanconianemia, amegakaryocytic thrombocytopenia, aplastic anemia, DiamondBlackfan anemia, dyskeratosis congenita, paroxysmal nocturnalhemoglobinuria (PNH), Pearson syndrome, Shwachman Diamond syndrome,thrombocytopenia, and myelodysplastic syndrome.

A disease or condition that is an age-related telomere shorteningdisease or a premature aging disease that may benefit from Zscan4+ humancells and/or an agent that increases Zscan4 expression in human cellsmay include, without limitation, Werner syndrome, Bloom's syndrome,Hutchinson-Gilford progeria syndrome, Cockayne syndrome, Xerodermapigmentosa, Ataxia telangiectasia, Rothmund Thomson syndrome,Trichothiodystrophy, Juberg-Marsidi syndrome, and Down syndrome.

In some embodiments, a subject of the present disclosure has a diseaseor condition associated with a chromosome abnormality. A chromosomeabnormality may refer to any anomaly, aberration, or mutation in achromosome that results in a missing, extra, or irregular portion ofchromosomal DNA. In certain embodiments, a chromosome abnormality mayresult in an atypical number of chromosomes or a structural abnormalityin one or more chromosomes. In certain embodiments, a chromosomeabnormality may include a karyotype abnormality, such as aneuploidy. Asused herein, aneuploidy may refer to an abnormal number of wholechromosomes or parts of chromosomes, including, without limitation,chromosome nullisomy, chromosome monosomy, chromosome trisomy,chromosome tetrasomy, and chromosome pentasomy. Examples of humananeuploidies include, without limitation, trisomy 21, trisomy 16,trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), monosomy X(Turner's syndrome), XXX aneuploidy, XXY aneuploidy, and XYY aneuploidy.Examples of human segmental aneuploidy include, without limitation, 1p36duplication, dup(17)(p11.2p11.2) syndrome, Pelizaeus-Merzbacher disease,dup(22)(q11.2q11.2) syndrome, and cat-eye syndrome. In some embodiments,an aneuploidy may include one or more deletions of sex or autosomalchromosomes, which can result in a condition such as Cri-du-chatsyndrome, Wolf-Hirschhorn, Williams-Beuren syndrome, Charcot-Marie-Toothdisease, Hereditary neuropathy with liability to pressure palsies,Smith-Magenis syndrome, Neurofibromatosis, Alagille syndrome,Velocardiofacial syndrome, DiGeorge syndrome, Steroid sulfatasedeficiency, Kallmann syndrome, Microphthalmia with linear skin defects,Adrenal hypoplasia, Glycerol kinase deficiency, Pelizaeus-Merzbacherdisease, Testis-determining factor on Y, Azoospermia (factor a),Azoospermia (factor b), Azoospermia (factor c), or 1p36 deletion.Accordingly, in certain embodiments, Zscan4+ human cells and/or an agentthat increases Zscan4 expression in human cells may be used to treataneuploidy or a disease, disorder, or condition associated with ananeuploidy.

Various types of diseases, disorders, and conditions may benefit fromZscan4+ human cells and/or an agent that increases Zscan4 expression inhuman cells including, without limitation, immunological deficiencies,an autoimmune disease, an autoimmune disorder, chronic ulcers,atherosclerosis, cancer, a neurologic injury, a degenerative disorder, aneurodegenerative disorder, wound healing, muscle repair, cardiac musclerepair, cartilage replacement, arthritis, osteoarthritis, toothregeneration, blindness, age-related blindness due to proliferativedecline of retinal pigmented epithelial cells, deafness, bone marrowfailure, bone marrow transplant, diabetes, muscular dystrophy, Duchennemuscular dystrophy, a genetic disease, a genetic mutation, and DNAdamage.

Cancers include malignant tumors that are characterized by abnormal oruncontrolled cell growth. Cancers are frequently associated with genomeinstability, chromosome abnormalities, DNA mutations, and aberranttelomere regulation. Based on the Zscan4 activities described herein,such as increasing the genome stability and correcting chromosomeabnormalities, Zscan4 biologics of the present disclosure (e.g., agentsof the present disclosure that increase Zscan4 expression in humancells) may be administered to treat cancer patients. As disclosed hereinin Example 17, applicant has shown that Zscan4 biologics can slow downthe proliferation of cancer cells. Accordingly, in some embodiments, anagent of the present disclosure that increases Zscan4 expression inhuman cells may be administered to prevent cancer cells from becomingmore aggressive due to genome instability, chromosome abnormalities, DNAmutations, and/or aberrant telomere regulation. Furthermore, Zscan4+human cells, such as immune cells, may be administered to cancerpatients to enhance their physical abilities to suppress the growth ofcancer cells. In other embodiments, Zscan4+ human cells may be used inpatients who have had a tumor removed, wherein specific cellsdifferentiated from Zscan4+ cells are used to reconstruct the removedtissues and/or organs. In other embodiments, an agent of the presentdisclosure that increases Zscan4 expression in human cells may beadministered to represses the growth (e.g., proliferation) of the cancercells.

It is known that human cancer tissues (e.g., tumors) contain cancer stemcells, which are not actively proliferating and are resistant to cancerchemotherapy (e.g., treatment with chemotherapeutic agents such ascisplatin). It is believed that cancer stem cells can survive treatmentwith chemotherapy, and thus results in the recurrence of the cancerafter the treatment. It is also believed that endogenous ZSCAN4expression occurs in certain cancer stem cells, thus providing the cellswith protection from the chemotherapeutic agents. As such, it isbelieved that agents that reduce the expression of endogenous ZSCAN4,such as siRNAs or shRNAs specific for ZSCAN4, may be administered tocancer patients who are undergoing or will undergo chemotherapy toreduce or eliminate resistance to one or more chemotherapeutic agents inthe cancer stem cells, thereby improving responsiveness to the one ormore chemotherapeutic agents in the patient.

Exemplary cancers that can benefit from Zscan4+ human cells and/or anagent that increases Zscan4 expression in human cells provided hereininclude but are not limited to cancers of the heart (e.g., sarcoma(angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma,rhabdomyoma, fibroma, lipoma and teratoma), lung (e.g., bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma);gastrointestinal tract (e.g., esophagus (squamous cell carcinoma,adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma), genitourinary tract (e.g., kidney (adenocarcinoma, Wilms'tumor, nephroblastoma, lymphoma, leukemia), bladder and urethra(squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma),prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma,embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma,interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors,lipoma), liver (e.g., hepatoma (hepatocellular carcinoma),cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellularadenoma, hemangioma), bone (e.g., osteogenic sarcoma (osteosarcoma),fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing'ssarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma,malignant giant cell tumor, chordoma, osteochondroma (osteocartilaginousexostoses), benign chondroma, chondroblastoma, chondromyxofibroma,osteoid osteoma and giant cell tumors), nervous system (e.g., skull(osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma >pinealoma!, glioblastomamultiforme, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma)),gynecological cancers (e.g., uterus (endometrial carcinoma), cervix(cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovariancarcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma,endometrioid tumors, Brenner tumor, clear cell carcinoma, unclassifiedcarcinoma, granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma,embryonal rhabdomyosarcoma, fallopian tubes (carcinoma)), hematologiccancers (e.g., blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma)), skin (e.g.,malignant melanoma, basal cell carcinoma, squamous cell carcinoma,Kaposi's sarcoma, moles, dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis), and adrenal glands (e.g.,neuroblastoma).

In some embodiments, a patient with an autoimmune disease is selectedfor treatment. An autoimmune disease may refer to a disease resultingfrom an aberrant immune response, such as the production of antibodiesor cytotoxic T cells specific for a self-antigen or a subject's owncells or tissues. Autoimmune diseases can result from an overactiveimmune response of the body against substances and tissues normallypresent in the body. In some examples, the autoimmune disease is berestricted to certain organs (e.g., in thyroiditis) or can involve aparticular tissue in different places (e.g., Goodpasture's disease whichmay affect the basement membrane in both the lung and the kidney).Accordingly, in some embodiments, Zscan4+ human cells, such ashematopoietic stem cells and/or immune cells, and/or an agent of thepresent disclosure that increases Zscan4 expression in human cells, maybe used to treat an autoimmune disease in a subject, by correcting theimmune system of a patient with an autoimmune disease. Exemplaryautoimmune diseases that can benefit from Zscan4+ human cells and/or anagent that increases Zscan4 expression in human cells provided hereininclude but are not limited to, rheumatoid arthritis, juvenileoligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis,Sjogren's syndrome, multiple sclerosis, experimental autoimmuneencephalomyelitis, inflammatory bowel disease (for example, Crohn'sdisease, ulcerative colitis), autoimmune gastric atrophy, pemphigusvulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes,myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosingcholangitis, sclerosing sialadenitis, systemic lupus erythematosis,autoimmune thrombocytopenia purpura, Goodpasture's syndrome, Addison'sdisease, systemic sclerosis, polymyositis, dermatomyositis, autoimmunehemolytic anemia, and pernicious anemia.

In some embodiments, the subject selected is one who has suffered aneurologic injury or suffers from a neurodegenerative disorder. Aneurological injury may refer to a trauma to the nervous system (such asto the brain or spinal cord or particular neurons), which adverselyaffects the movement and/or memory of the injured patient. For example,such patients may suffer from dysarthria (a motor speech disorder),hemiparesis or hemiplegia. Neurologic injuries can result from a traumato the nervous system (such as to the brain or spinal cord or particularneurons), which adversely affects the movement and/or memory of theinjured patient. Such traumas may be caused by an infectious agent(e.g., a bacterium or virus), a toxin, an injury due to a fall or othertype of accident, or genetic disorder, or for other unknown reasons.Accordingly, in some embodiments, Zscan4+ human cells, such ashematopoietic stem cells, neural stem cells, mesenchymal stem cells,and/or immune cells, and/or an agent of the present disclosure thatincreases Zscan4 expression in human cells, may be used to treat aneurologic injury in a subject, by rejuvenating tissue stem cells in thenervous system of a patient that has suffered a neurologic injury, whererejuvenating tissue stem cells in the nervous system produces neuronsand glial cells, thereby repairing defects in nervous system. In someembodiments, the patient may have suffered a neurologic injury, such asa brain or spinal cord injury resulting from an accident, such as anautomobile or diving accident, or from a stroke.

A neurodegenerative disease is a condition in which cells of the brainand spinal cord are lost. Neurodegenerative diseases result fromdeterioration of neurons or their myelin sheath which over time lead todysfunction and disabilities. Conditions that result can cause problemswith movement (such as ataxia) and with memory (such as dementia).Accordingly, in some embodiments, Zscan4+ human cells, such ashematopoietic stem cells, neural stem cells, mesenchymal stem cells,and/or immune cells, and/or an agent of the present disclosure thatincreases Zscan4 expression in human cells, may be used to treat aneurodegenerative disease in a subject, by rejuvenating tissue stemcells in the nervous system of a patient suffering from aneurodegenerative disease, where rejuvenating tissue stem cells in thenervous system produces neurons and glial cells, thereby repairingdefects in nervous system. In some embodiments, the Zscan4+ human cellsand/or agent rejuvenate the nervous system of the subject and revert thedegenerative conditions of the disease. Exemplary neurodegenerativediseases Zscan4+ human cells and/or an agent that increases Zscan4expression in human cells provided herein include but are not limitedto: adrenoleukodystrophy (ALD), alcoholism, Alexander's disease, Alper'sdisease, Alzheimer's disease, amyotrophic lateral sclerosis (LouGehrig's Disease), ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), bovine spongiformencephalopathy (BSE), Canavan disease, cerebral palsy, Cockaynesyndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, familialfatal insomnia, frontotemporal lobar degeneration, Huntington's disease,HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy bodydementia, neuroborreliosis, Machado-Joseph disease (Spinocerebellarataxia type 3), Multiple System Atrophy, multiple sclerosis, narcolepsy,Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease,Pick's disease, primary lateral sclerosis, prion diseases, progressivesupranuclear palsy, Refsum's disease, Sandhoff disease, Schilder'sdisease, subacute combined degeneration of spinal cord secondary toPernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease (also knownas Batten disease), spinocerebellar ataxia, spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, toxicencephalopathy.

Zscan4+ human cells can be obtained or generated using the methodsdescribed herein. Methods of administering Zscan4+ human cells tomammalian subjects are known in the art. For example, Zscan4+ humancells administered to a subject in need of such therapy via injection,such as subcutaneous, intramuscular, intradermal, intraperitoneal,intravenous or intra-arterial administration. In some embodiments,Zscan4+ human cells are administered directly to the area in need oftreatment, such as to a cancerous organ or tissue, or to the brain orspinal cord. In some embodiments, Zscan4+ human cells are administeredalone, in the presence of a pharmaceutically acceptable carrier (such asencapsulated in a suitable polymer) or in the presence of othertherapeutic agents. In some embodiments, a subject is administered atleast 20,000 Zscan4+ human cells, such as at least 50,000, at least100,000, at least 500,000, at least 1,000,000, or at least 2,000,000Zscan4+ human cells.

In some aspects, the methods of the present disclosure involve the useof a therapeutic amount of an agent that increases expression of Zscan4.A therapeutic amount of an agent may refer to the amount of atherapeutic agent sufficient to achieve the intended purpose. Forexample, a therapeutic amount of Zscan4+ human cells and/or an agentthat increases Zscan4 expression in a human cell to treat a disease orcondition associated with a telomere abnormality is an amount sufficientto reduce the disease or condition or one or more symptoms of thedisease or condition. A therapeutic amount may in some example not treatthe disease or condition or symptoms of the disease or condition 100%.However, a decrease in any known feature or symptom of the disease orcondition, such as a decrease of at least 10%, at least 15%, at least25%, at least 30%, at least 50%, at least 60%, at least 70%, at least75%, at least 85%, at least 95%, or greater, can be therapeutic. Thetherapeutic amount of a given therapeutic agent will vary with factorssuch as the nature of the agent, the route of administration, the sizeand/or age of the subject to receive the therapeutic agent, and thepurpose of the administration. The therapeutic amount in each individualcase can be determined empirically without undue experimentation by askilled artisan according to established methods in the art.

In some aspects, the methods of the present disclosure involve the useof a pharmaceutical agent. A pharmaceutical agent may refer to achemical compound, small molecule, or other composition, such as aZscan4+ human cell, capable of inducing a desired therapeutic orprophylactic effect when properly administered to a subject or a cell.“Incubating” includes a sufficient amount of time for a drug to interactwith a cell. “Contacting” includes incubating a drug in solid or inliquid form with a cell.

The pharmaceutically acceptable carriers of use in the presentdisclosure are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the Zscan4 proteins, Zscan4 nucleic acid molecules,retinoids, agents that induce oxidative stress, and cells disclosedherein. In general, the nature of the carrier will depend on theparticular mode of administration being employed. For instance,parenteral formulations usually comprise injectable fluids that includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, aqueous dextrose,glycerol or the like as a vehicle. For solid compositions (e.g., powder,pill, tablet, or capsule forms), conventional non-toxic solid carrierscan include, for example, pharmaceutical grades of mannitol, lactose,starch, or magnesium stearate. In addition to biologically-neutralcarriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example, sodium acetate or sorbitan monolaurate.

In one example, Zscan4+ human cells and/or an agent that increasesZscan4 expression in human cells are encapsulated into a semipermeablepolymer membrane and the polymer membrane transplanted into a tissuesite of a host subject. Such methods may achieve local, long-termchronic delivery of a therapeutic substance with the capability ofregulating release of the substance. See U.S. Pat. No. 5,573,528 fordescription of encapsulation of compounds and cells. In one embodiment,Zscan4+ human cells and/or an agent that increases Zscan4 expression inhuman cells are encapsulated within a polymer membrane. The encapsulatedpolymer membrane is then transplanted into a tissue site of a hostsubject. In one embodiment, the tissue site is central nervous system,such as brain or spinal cord.

The semipermeable polymer membrane can be synthetic or natural. Examplesof polymer that can be used include polyethersulfone (PES),polyacrylonitrile-co-vinyl chloride (P[AN/VC], poly(lactic acid),poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid,collagen, and the like. Delivery of encapsulated Zscan4+ human cellsand/or an agent that increases Zscan4 expression in human cells within apolymer membrane can avoid host rejection and immune response to cells,and problems associated with rejection and inflammation. In addition,cells contained within the polymer membrane are shielded by the wall ofthe polymer (i.e., the walls of the individual fibers, fibrils, films,sprays, droplets, particles, etc.) from immune surveillance while stillmaintaining cell viability and allowing transport of molecules,nutrients and metabolic products through the polymer walls. The graftingof polymer-encapsulated cells has been developed by Aebischer et al.,1991, Transplant, 111:269-275, and has been successfully used with bothnon-human primates and humans (Aebischer et al., 1994, Transplant,58:1275-1277). See also U.S. Pat. No. 6,110,902.

In one embodiment, Zscan4+ human cells are encapsulated by firstembedding them into a matrix of either collagen, agarose or PVA(polyvinylalcohol). Subsequently, the embedded cells are injected intohollow fibers made of polypropylene of a 60:40 copolymer ofpolyacrylonitrile:polyvinylchloride. The fibers are cut into pieces andend-sealed for implantation. In one embodiment, the encapsulated cellshave about 20,000 to about 2,000,000 Zscan4+ human cells.

In some examples, the Zscan4+ human cells are of exogenous origin.Exogenous cells may refer to cells obtained from sources other than thesubject in which they are implanted for treatment. Exogenous cells canbe from other organisms of the same species (such as human-derived cellsfor use in a human patient). Exogenous cells can also be fromheterologous sources, i.e., from a species distinct from the subject tobe therapeutically treated (such as mouse cells for use in a human).Zscan4+ human cells can also be taken from an isogenic source, i.e.,from the subject who is to receive the cells. After harvesting the cellsfrom the subject, the cells can be genetically modified (e.g., a nucleicacid encoding Zscan4 is introduced) or selected/enriched for Zscan4+human cells, then re-implanted back to the subject. Since the cells areisogeneic, no immune response is expected.

In one aspect, the Zscan4+ human cells are immortalized. For example andnot by way of limitation, cells can be conditionally immortalized (suchthat the cells grow well in tissue culture at reduced temperatures, yetdiscontinue division once implanted into a patient and maintained at 37°C.) or constitutively immortalized (e.g., transfection with constructsexpressing large T antigen, or immortalization by Epstein Barr virus) bymethods well known in the art. Another method of delivering Zscan4+human cells into a host subject is to directly transplant the cells intothe target area of a tissue site. Once transplanted, these cellssurvive, migrate and integrate seamlessly into the host tissue. In oneembodiment, the Zscan4+ human cells are directly transplanted into thenervous system of the host subject, such as a developing nervous systemor a nervous system that has suffered a trauma or in a subject having aneurological disorder. When transplanted into a developing nervoussystem, the Zscan4+ human cells will participate in processes of normaldevelopment and will respond to the host's developmental cues. Thetransplanted neural precursor cells will migrate along establishedmigratory pathways, will spread widely into disseminated areas of thenervous system and will differentiate in a temporally and regionallyappropriate manner into progeny from both the neuronal and gliallineages in concert with the host developmental program. Thetransplanted Zscan4+ human cell is capable of non-disruptiveintermingling with the host neural precursor cells as well asdifferentiated cells. The transplanted cells can replace specificdeficient neuronal or glial cell populations, restore defectivefunctions and can express foreign genes in a wide distribution.

The Zscan4+ human cells can also be transplanted into a developednervous system. The transplanted neural precursor cells can form astable graft, migrate within the host nervous system, intermingle andinteract with the host neural progenitors and differentiated cells. Theycan replace specific deficient neuronal or glial cell populations,restore deficient functions and activate regenerative and healingprocesses in the host's nervous system. In one embodiment, the stablegraft is a graft established in the central nervous system or theperipheral nervous system.

Similar methods can be used to directly transplant Zscan4+ human cellsinto any region in need of human cell therapy. Such cells may beundifferentiated or differentiated into the desired cell type in vitro(then administered to a subject in need thereof). For example, whereorgan regeneration is desired, for example for replacement of organs ortissues removed to treat cancer or lost for other reasons (e.g., teeth,hair, cells of the ear or eyes, skin or muscle). In one embodiment,Zscan4+ human cells are directly transplanted into the heart, forexample to regenerate cardiac tissue or cells lost to myocardialinfarction. In one embodiment, Zscan4+ human cells are directlytransplanted into the pancreas, for example to regenerate cells in asubject with diabetes. In one embodiment, Zscan4+ human cells aredirectly transplanted into the bone or administered systemically, forexample to regenerate bone marrow cells in a subject having cancer.

The therapeutic dose and regimen most appropriate for patient treatmentwill vary with diseases or conditions to be treated, and according tothe patient's weight and other parameters. An effective dosage andtreatment protocol can be determined by conventional means, startingwith a low dose in laboratory animals and then increasing the dosagewhile monitoring the effects, and systematically varying the dosageregimen. Numerous factors can be taken into consideration by a clinicianwhen determining an optimal dosage for a given subject. Factors includethe size of the patient, the age of the patient, the general conditionof the patient, the particular disease being treated, the severity ofthe disease, the presence of other drugs in the patient, and the like.The trial dosages would be chosen after consideration of the results ofanimal studies and the clinical literature.

Accordingly, Zscan4+ human cells and/or an agent that increases Zscan4expression in human cells are administered to subjects so as to reduceor ameliorate symptoms associated with a particular disorder,particularly those associated with telomere abnormalities. Therapeuticendpoints for the treatment of cancer can include a reduction in thesize or volume of a tumor, reduction in angiogenesis to the tumor, orreduction in metastasis of the tumor. If the tumor has been removed,another therapeutic endpoint can be regeneration of the tissue or organremoved. Effectiveness of cancer treatment can be measured using methodsin the art, for example imaging of the tumor or detecting tumor markersor other indicators of the presence of the cancer. Therapeutic endpointsfor the treatment of autoimmune diseases can include a reduction in theautoimmune response. Effectiveness of autoimmune disease treatment canbe measured using methods in the art, for example measuring ofautoimmune antibodies, wherein a reduction in such antibodies in thetreated subject indicates that the therapy is successful. Therapeuticendpoints for the treatment of neurodegenerative disorders can include areduction in neurodegenerative-related deficits, e.g., an increase inmotor, memory or behavioral deficits. Effectiveness of treatingneurodegenerative disorders can be measured using methods in the art,for example by measuring cognitive impairment, wherein a reduction insuch impairment in the treated subject indicates that the therapy issuccessful. Therapeutic endpoints for the treatment of neurologicinjuries can include a reduction in injury-related deficits, e.g., anincrease in motor, memory or behavioral deficits. Effectiveness oftreating neurologic injuries can be measured using methods in the art,for example by measuring mobility and flexibility, wherein an increasein such in the treated subject indicates that the therapy is successful.Treatment does not require 100% effectiveness. A reduction in thedisease (or symptoms thereof) of at least about 10%, about 15%, about25%, about 40%, about 50%, or greater, for example relative to theabsence of treatment with Zscan4+ human cells and/or an agent thatincreases Zscan4 expression in human cells, is considered effective.

In some examples, Zscan4+ human cells are administered at a dose fromabout 1×10⁴ cells to about 1×10⁷ cells in a mouse or other small mammal,or a dose from about 1×10⁴ cells to about 1×10¹⁰ cells in a human orother large mammal. In one specific, non-limiting embodiment, atherapeutically effective amount is about 1×10⁶ cells. Other therapeuticagents (for example, chemical compounds, small molecules, or peptides)can be administered in a therapeutically effective dose in combinationwith the Zscan4+ human cells (for example shortly before or after, orsimultaneously) in order to achieve a desired effect in a subject beingtreated. An effective amount of Zscan4+ human cells may be administeredin a single dose, or in several doses, for example daily, during acourse of treatment. However, one skilled in the art will appreciatethat the effective amount of Zscan4+ human cells will be dependent onthe agent applied, the subject being treated, the severity and type ofthe affliction, and the manner of administration of the agent.

Agents of the present disclosure that increase expression of Zscan4 mayalso be used to rejuvenate skin, treat atopic dermatitis, or skinlesions in a subject in need thereof, by, for example, applying an agentof the present disclosure that increase expression of Zscan4 to the skinof the subject.

Agents of the present disclosure that increase expression of Zscan4 mayalso be used to treat hair loss by stimulating hair growth in a subjectin need thereof, by, for example, applying an agent of the presentdisclosure that increase expression of Zscan4 to the scalp of thesubject. Agents of the present disclosure that increase expression ofZscan4 may also be used to prevent or treat hair graying in a subject inneed thereof by applying an agent of the present disclosure thatincrease expression of Zscan4 to the scalp to increasing telomere lengthand/or genome stability melanocyte stem cells in hair follicles, whosedysfunction causes gray hair.

As disclosed herein, limbal stem cells regenerate corneas, and as such,it is believed that increasing telomere length and/or genome stabilityin limbal stem cells by increasing expression of Zscan4 would rejuvenatecorneas in a subject in need thereof. Increasing expression of Zscan4 incorneas may also be used to treat dry eyes in a subject in need thereof.Accordingly, agents of the present disclosure that increase expressionof Zscan4 may also be used to rejuvenate corneas and/or treat dry eye ina subject in need thereof, by, for example, applying an agent of thepresent disclosure that increase expression of Zscan4 to a cornea of thesubject.

As disclosed herein, idiopathic pulmonary fibrosis is known to be causedby the telomere shortening. Accordingly, agents of the presentdisclosure that increase expression of Zscan4 may also be used to treatidiopathic pulmonary fibrosis in a subject in need thereof, by, forexample, formulating an agent of the present disclosure that increasesexpression of Zscan4 (e.g., a Zscan4 polynucleotide of the presentdisclosure) such that it can be inhaled by the subject in order to treatthe idiopathic pulmonary fibrosis.

Agents of the present disclosure that increase expression of Zscan4 mayalso be used to treat atherosclerosis and/or a coronary heart disease ina subject in need thereof, by, for example, administering an agent ofthe present disclosure to the bloodstream of the subject such that theagent contacts and increases telomere length and/or genome stability ofvascular endothelial cells, thereby treating atherosclerosis and/or acoronary heart disease in the subject.

Agents of the present disclosure that increase expression of Zscan4 mayalso be used to provide resistance to one or more genotoxic agents inone or more human cells and/or a subject in need thereof, by, forexample, contacting the one or more human cells with an agent of thepresent disclosure that increases expression of Zscan4(e.g., a Zscan4polynucleotide of the present disclosure) or administering such an agentto a subject in need thereof, such that increasing expression of Zscan4increases resistance to one or more genotoxic agents in the one or morehuman cells or subject. Advantageously, Zscan4 expression can be used toprovide resistance for any known genotoxic agent or drug, including,without limitation, mitomycin C and cisplatin.

It is known that there are regions in the genome of iPS cells where DNAmethylation patterns differ from those of human embryonic stem (ES)cells, which is believed to cause problems in the iPS cells (e.g., seeOhi, Yet al., Nat Cell Biol. 2011 May; 13(5):541-9). Without wishing tobe bound by theory, it is believed that agents of the present disclosurethat increase expression of Zscan4 will improve iPS cells by inducing aDNA methylation pattern in human iPS cells that is more similar to thatof human ES cells. It is further believed that inducing a more human EScell-like DNA methylation pattern in human iPS can may such cells saferfor therapeutic use. Accordingly, in certain embodiments, agents of thepresent disclosure that increase expression of Zscan4 may be used toinduce a human embryonic stem cell-like DNA methylation pattern in oneor more human induced pluripotent stem (iPS) cells, by, for example,contacting one or more human iPS cells with an agent of the presentdisclosure that increases expression of Zscan4(e.g., a Zscan4polynucleotide of the present disclosure), such that increasingexpression of Zscan4 induces a human embryonic stem cell-like DNAmethylation pattern in the one or more human iPS cells.

Agents of the present disclosure that increase expression of Zscan4 mayalso be used to rejuvenate aged oocytes and correct karyotypeabnormalities in both oocyte cells and in vitro fertilized oocytes, suchas zygotes or preimplantation embryos between the one-cell stage and theblastocyst stage to increase the success rate of in vitro fertilization(IVF); to increase the success rate of healthy full-term pregnancies in,for example, older women; to correct karyotype abnormalities, such asaneuploidy; and to reduce the risk of developmental disorders, such asDown syndrome. Such treatments may be of particular use for in vitrofertilization (IVF) and in IVF clinics. Accordingly, in certainembodiments, agents of the present disclosure that increase expressionof Zscan4 may be used to rejuvenate one or more human oocyte cells,increase genome stability in one or more human oocyte cells, and/orcorrect one or more karyotype abnormalities in one or more human oocytecells by, for example, contacting one or more human oocyte cells with anagent of the present disclosure that increases expression of Zscan4(e.g., a Zscan4 polynucleotide of the present disclosure), such thatincreasing expression of Zscan4 rejuvenates the one or more human oocytecells, increases genome stability in the one or more human oocyte cells,and/or corrects one or more karyotype abnormalities in the one or morehuman oocyte cells. In some embodiments, the one or more human oocytecells are isolated from a subject prior to contacting with the agentthat increases expression of Zscan4. In other embodiments, once the oneor more human oocyte cells have been treated with the agent thatincreases expression of Zscan4, the cells undergo in vitrofertilization.

In other embodiments, agents of the present disclosure that increaseexpression of Zscan4 may be used to increase genome stability and/orcorrect one of more karyotype abnormalities in one or more fertilizedhuman oocytes by, for example, contacting the one or more fertilizedhuman oocytes with an agent of the present disclosure that increasesexpression of Zscan4 (e.g., a Zscan4 polynucleotide of the presentdisclosure), such that increasing expression of Zscan4 increases genomestability and/or corrects the one or more karyotype abnormalities in theone or more fertilized human oocytes. In some embodiments, the one ormore fertilized human oocytes were fertilized by in vitro fertilizationprior to being contacted with agent that increases expression of Zscan4.In other embodiments, the one or more human oocytes are isolated from asubject prior to being fertilized. In yet other embodiments, the one ormore fertilized oocytes are preimplantation embryos between the one-cellstage and the blastocyst stage.

In certain embodiments, the agent of the present disclosure thatincreases expression of Zscan4 corrects one or more karyotypeabnormalities that include, without limitation, an aneuploidy, such astrisomy 21 (Down syndrome), trisomy 16, trisomy 18 (Edwards syndrome),trisomy 13 (Patau syndrome), monosomy X (Turner's syndrome), XXXaneuploidy, XXY aneuploidy, and XYY aneuploidy; a segmental aneuploidy,such as 1p36 duplication, dup(17)(p11.2p11.2) syndrome,Pelizaeus-Merzbacher disease, dup(22)(q11.2q11.2) syndrome, and cat-eyesyndrome; and a condition such as Cri-du-chat syndrome, Wolf-Hirschhorn,Williams-Beuren syndrome, Charcot-Marie-Tooth disease, Hereditaryneuropathy with liability to pressure palsies, Smith-Magenis syndrome,Neurofibromatosis, Alagille syndrome, Velocardiofacial syndrome,DiGeorge syndrome, Steroid sulfatase deficiency, Kallmann syndrome,Microphthalmia with linear skin defects, Adrenal hypoplasia, Glycerolkinase deficiency, Pelizaeus-Merzbacher disease, Testis-determiningfactor on Y, Azoospermia (factor a), Azoospermia (factor b), Azoospermia(factor c), and 1p36 deletion.

As disclosed herein, the human genes SERPINB4, DNMT3L, and DUX4 aremarker genes, whose expression is unregulated when Zscan4 gene isexpressed in human cells. As such, it is believed that SERPINB4, DNMT3L,and DUX4 may be used as markers for the effects of Zscan4 in humancells. Accordingly, in some embodiments methods are provided fordetermining one or more Zscan4-induced effects in one or more humancells, for example, by contacting the one or more human cells with anagent that increases expression of Zscan4 in one or more human cells;measuring expression levels of SERPINB4, DNMT3L, and/or DUX4 in the oneor more human cells; and comparing the expression levels of SERPINB4,DNMT3L, and/or DUX4 in the one or more human cells to the expressionlevels of SERPINB4, DNMT3L, and/or DUX4 in one or more correspondinghuman cells that are not contacted with the agent, where an increase inthe expression levels of SERPINB4, DNMT3L, and/or DUX4 in the one ormore human cells indicates the presence of one or more Zscan4-inducedeffects in the one or more human cell.

In some embodiments, the subjects of the present disclosure arenon-human animals. Non-human animals may refer to all animals other thanhumans. A non-human animal includes, but is not limited to, a non-humanprimate, a farm animal such as swine, cattle, and poultry, a sportanimal or pet such as dogs, cats, horses, hamsters, rodents, such asmice, or a zoo animal such as lions, tigers or bears. In one embodiment,the non-human animal is a mouse.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1: Zscan4 Expression Corrects Chromosome Abnormalitiesin Mouse Embryonic Stem Cells

This example describes the finding that expression of Zscan4, either bya synthetic mRNA encoding Zscan4 or a Sendai virus vector expressingZscan4, can correct chromosome abnormalities in mouse embryonic stem(ES). This example also demonstrates that synthetic mRNAs encodingZscan4 and Sendai virus vector expressing Zscan4 can be used astherapeutic biologics.

Materials and Methods Cell Culture

The MC1ZE mouse ES cell line was previously reported (Amano et al., NatCommun, 2013; 4:1966.). MC1ZE cells were used as typical mouse ES cellswhich show poor karyotypes (i.e., only about 20% of cells carryeuploidy) due to long-term cultures (passage number 33), and theintegration of an exogenous gene. Cells were cultured at 37° C. in 5%CO2 in the complete ES medium as previously described (Amano et al., NatCommun, 2013; 4:1966.): DMEM (Gibco), 15% FBS (Atlanta Biologicals),1000 U/ml leukemia inhibitory factor (LIF) (ESGRO, Chemicon), 1 mMsodium pyruvate, 0.1 mM non-essential amino acids (NEAA), 2 mM GlutaMAX,0.1 mM beta-mercaptoethanol, and penicillin/streptomycin (50 U/50μg/ml). Medium was changed daily and cells were passaged every 2 to 3days routinely.

Synthetic mRNA

For synthesis of modified mRNA, mRNA synthesis was performed as reportedpreviously by Warren et al. (Warren et al., Cell Stem Cell, 2010 Nov. 5;7(5):618-30). Using these protocol from Warren et al., mRNAs weresynthesized by in vitro transcription of template DNAs encoding humanZSCAN4 or green fluorescent protein (GFP) with mixtures of modifieddNTPs to increase RNA stability as well as translation efficiency inmammalian cells. The following modified dNTPs were used:3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, 5-methylcytidine triphosphate,and pseudouridine triphosphate.

Sendai Virus Vectors

Sendai vectors that express either mouse Zscan4c (SeV18+mZscan4/ΔF) orhuman ZSCAN4 (SeV18+hZSCAN4/ΔF) were custom-made by MBL (Medical &Biological Laboratories Co, LTD). These vectors are referred to as“SeVmZscan4” or “SeVhZSCAN4”, respectively, herein. As a control, thesame Sendai vector was used, but the vector expressed a greenfluorescent protein variant, rather than Zscan4. These Sendai vectorslack the F protein, and thus, are not transmissible (Inoue et al., JVirol. 77: 23238-3246, 2003).

Sendai vectors that express either mouse Zscan4c fused to aTamoxifen-controllable ERT2 domain (SeV18+mZERT2/ΔF), or human ZSCAN4fused to Tamoxifen-controllable ERT2 domain (SeV18+hZERT2/ΔF) werecustom-made by MBL (Medical & Biological Laboratories Co, LTD). Thesevectors are referred to as “SeVmZERT2” or “SeVhZERT2”, respectively,herein. These Sendai vectors also lack the F protein, and thus, are nottransmissible (Inoue et al., J Virol. 77: 23238-3246, 2003).

Additionally, temperature-sensitive Sendai vectors that express eithermouse Zscan4 (SeV18+mZscan4/TS15ΔF) or human ZSCAN4(SeV18+hZSCAN4/TS15ΔF) were custom-made by MBL (Medical & BiologicalLaboratories Co, LTD). These vectors are referred to as“SeVmZscan4-TS15” or “SeVhZSCAN4-TS15”, respectively, herein. TheseSendai vectors are functional at 35° C., and inactive at 37° C. (Ban etal., Proc Natl Acad Sci USA. 2011; 108(34):14234-14239). As a control,the same Sendai vector was used, but the vector expressed a greenfluorescent protein variant, rather than Zscan4. This vector is referredto as “SeVAG-TS15” herein. These Sendai vectors also lacks the Fprotein, and thus, it is not transmissible (Inoue et al., J Virol. 77:23238-3246, 2003).

Karyotype Analysis

Karyotype analysis was performed by G-banding carried out as describedin Amano et al., Nat Commun, 2013; 4:1966.

Results Synthetic mRNAs Encoding Mouse or Human ZSCAN4 CorrectChromosome Abnormalities in Mouse Embryonic Stem Cells

FIG. 1A shows the experimental procedure: MC1ZE mouse ES cells (atpassage 33) were plated in a 6 well dish at a concentration of 5×10⁴cells/well and transfected with either 1 of synthetic GFP mRNAs(control) or 1 μg of synthetic human ZSCAN4 mRNAs using 5 μl ofLipofectamine (RNAiMAX: Life Technologies, California, USA). In additionto cells transfected with GFP mRNAs, non-transfected cells were alsoused as controls. Cells were passaged every 2-3 days, followed by thetransfection with synthetic mRNAs. Karyotyping of cells was carried outat passage 33 (3 days after one-time transfection), at passage 34 (3days after three-time transfection), at passage 36 (3 days after 4-timetransfection), and at passage 40 (3 days after seven-time transfection).

As shown in FIG. 1B, the transfection of mouse ES cells with syntheticmRNAs of human ZSCAN4 corrected chromosome abnormalities and increasedthe number of cells with a normal karyotype (euploidy). Although theone-time transfection was sufficient to see a significant level ofcorrection, repeated transfections (e.g., seven times) further increasedthe number of cells with euploidy from around 20% to 40%. These resultsindicate that the introduction of human ZSCAN4 mRNAs into cells cancorrect abnormalities in chromosome numbers.

Sendai Virus Vectors Expressing Human ZSCAN4 Correct ChromosomeAbnormalities in Mouse Embryonic Stem Cells

FIG. 2A shows a summary of the karyotype analysis after treating mouseES cells with a Sendai virus vector expressing either mouse Zscan4c orhuman ZSCAN4. MC1ZE mouse ES cells (at passage 33 or 34) were plated ina 6 well dish at a concentration of; 5×10⁴ cells/well and treated witheither SeVAG (control), SeVmZscan4, or SeVhZSCAN4 at the MOI indicatedin FIG. 2A. As an additional control, MC1ZE cells without any treatmentwere used. After 3 days, the karyotype of cells was analyzed. As shownin FIG. 2A, the karyotype of mouse ES cells can be dramatically improvedby contacting with SeVmZscan4 or SeVhZSCAN4. Interestingly, the resultsshow that human ZSCAN4 worked better than mouse Zscan4c.

FIG. 2B shows a summary of karyotype analysis after treating mouse MC1ZEES cells with a Sendai virus vector expressing either a mouseZscan4c-ERT2 fusion protein or a human ZSCAN4-ERT2 fusion protein. It isknown that the protein fused with the ERT2 domain can be activated bythe presence of Tamoxifen (Tmx) in the cell culture medium. MC1ZE mouseES cells (at passage 33 or 34) were plated in a 6 well dish at aconcentration of 5×10⁴ cells/well and treated with either SeVmZERT2 orSeVhZERT2 at the MOI indicated in FIG. 2B. As a control, MC1ZE cellswithout any treatment were used. As shown in FIG. 2B, treatment witheither SeVmZERT2 or SeVhZERT2 can correct chromosome abnormalities evenwithout Tmx. Addition of Tmx, however, further enhanced the ability ofSeVmZERT2 and SeVhZERT2 to correct chromosome abnormalities. The resultsalso show that human ZSCAN4 works better than mouse Zscan4c: the fusionprotein, human ZSCAN4-ERT2, in the presence of Tmx, increased the numberof cells with euploidy from 15% to 53%.

FIG. 3 shows a summary of karyotype analysis after treating MC1ZE mouseES cells with a temperature-sensitive Sendai virus vector expressingeither mouse Zscan4c or human ZSCAN4. MC1ZE mouse ES cells (at passage33 or 34) were plated in a 6 well dish at a concentration of 5×10⁴cells/well and treated with either SeVAG-TS15 (control),SeVmZscan4-TS15, or SeVhZSCAN4-TS15 at the MOI indicated in FIG. 3. Asan additional control, MC1ZE cells without any treatment were used.After contacting the cells with these vectors, cells were cultured at35° C. for three days (FIG. 3A), at 35° C. for six days (FIG. 3B), andat 35° C. for three days, followed by culturing at 37° C. for three days(FIG. 3C). The correction of chromosome abnormalities by contactingcells with either SeVmZscan4-TS15 or SeVhZSCAN4-TS15 was observed in allthree conditions. Surprisingly, human ZSCAN4 was found to work betterthan mouse Zscan4c at correcting chromosome abnormalities. Thus, theseresults demonstrate that even in mouse cells, human ZSCAN4 producessurprisingly superior results over mouse Zscan4c. Indeed, the bestresult was obtained by treating cells with human ZSCAN4 (i.e.,SeVhZSCAN4-TS15) and culturing cells at 35° C. for three days, followedby the culture at 37° C. for three days: the treatment increased thenumber of cells with euploidy from 19% to 53%. In these experiments,culturing cells treated with the temperature-sensitive Sendai vectors atthe permissive temperature of 35° C. for three days followed byculturing at the inactivating temperature of 37° C. for three daysrepresents transient expression of Zscan4. In contrast culturing cellstreated with the temperature-sensitive Sendai vectors at the permissivetemperature of 35° C. for the full six days represents continuous Zscan4expression. Based on these conditions, the results indicate thetransient expression of Zscan4 works better than continuous expressionof Zscan4 at correcting chromosome abnormalities.

Example 2: Effects of Zscan4 Biologics on Mouse Embryonic Stem Cells

It has been shown previously that the forced expression of exogenousmouse Zscan4c from a plasmid vector integrated into the mouse genomeinduces the expression of a unique set of genes, including Tmem92,Stra8, and endogenous Zscan4 genes (Amano et al., Nat Commun, 2013;4:1966.).

This example demonstrates that Zscan4 biologics (e.g., expression ofZscan4, either by a synthetic mRNA encoding Zscan4 or a Sendai virusvector expressing Zscan4) exert the same function in mouse embryonicstem (ES) cells.

Materials and Methods Cell Culture

The MC1 mouse embryonic stem (ES) cell line was previously used astypical mouse pluripotent stem cells to demonstrate the function ofexogenously introduced Zscan4c gene, which was integrated into the mousegenome (Amano et al., Nat Commun, 2013; 4:1966.). MC1 cells werecultured at 37° C. in 5% CO2 in complete ES medium, as previouslydescribed (Amano et al., Nat Commun, 2013; 4:1966.): DMEM (Gibco), 15%FBS (Atlanta Biologicals), 1000 U/ml leukemia inhibitory factor (LIF)(ESGRO, Chemicon), 1 mM sodium pyruvate, 0.1 mM non-essential aminoacids (NEAA), 2 mM GlutaMAX, 0.1 mM beta-mercaptoethanol, andpenicillin/streptomycin (50 U/50μg/ml). The medium was changed daily andcells were passaged every 2 to 3 days routinely.

Synthetic mRNA

For synthesis of modified mRNA, mRNA synthesis was performed as reportedpreviously by Warren et al. (Warren et al., Cell Stem Cell, 2010 Nov. 5;7(5):618-30). Using these protocol from Warren et al., mRNAs weresynthesized by in vitro transcription of template DNAs encoding humanZSCAN4 or green fluorescent protein (GFP) with mixtures of modifieddNTPs to increase RNA stability as well as translation efficiency inmammalian cells. The following modified dNTPs were used:3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, 5-methylcytidine triphosphate,and pseudouridine triphosphate.

Sendai Virus Vectors

Sendai vectors that express either mouse Zscan4c (SeV18+mZscan4/ΔF) orhuman ZSCAN4 (SeV18+hZSCAN4/ΔF) were custom-made by MBL (Medical &Biological Laboratories Co, LTD). These vectors are referred to as“SeVmZscan4” or “SeVhZSCAN4”, respectively, herein. As a control, thesame Sendai was used, but the vector expressed a green fluorescentprotein variant rather than Zscan4. These Sendai vectors lack the Fprotein, and thus, are not transmissible (Inoue et al., J Virol. 77:23238-3246, 2003).

Sendai vectors that express either mouse Zscan4c fused to aTamoxifen-controllable ERT2 domain (SeV18+mZERT2/ΔF), or human ZSCAN4fused to Tamoxifen-controllable ERT2 domain (SeV18+hZERT2/ΔF) werecustom-made by MBL (Medical & Biological Laboratories Co, LTD). Thesevectors are referred to as “SeVmZERT2” or “SeVhZERT2”, respectively,herein. These Sendai vectors also lack the F protein, and thus, are nottransmissible (Inoue et al., J Virol. 77: 23238-3246, 2003).

Additionally, temperature-sensitive Sendai vectors that express eithermouse Zscan4 (SeV18+mZscan4/TS15ΔF) or human ZSCAN4(SeV18+hZSCAN4/TS15ΔF) were custom-made by MBL (Medical & BiologicalLaboratories Co, LTD). These vectors are referred to as“SeVmZscan4-TS15” or “SeVhZSCAN4-TS15”, respectively, herein. TheseSendai vectors are functional at 35° C., and inactive at 37° C. (Ban etal., Proc Natl Acad Sci USA. 2011; 108(34):14234-14239). As a control,the same temperature-sensitive Sendai vector was used, but the vectorexpressed a green fluorescent protein variant rather than Zscan4. Thisvector is referred to as “SeVAG-TS15” herein. These Sendai vectors alsolack the F protein, and thus, it is not transmissible (Inoue et al., JVirol. 77: 23238-3246, 2003).

Results

FIG. 4A shows the results of qRT-PCR analysis, monitoring the expressionlevels of Tmem92, Stra8, Actb (beta-actin control), and endogenousZscan4 genes after transfection of MC1mouse ES cells with syntheticmRNAs. Compared to control cells transfected with GFP mRNAs, humanZSCAN4 mRNAs increased the expression of the endogenous mouse Zscan4gene.

FIG. 4B shows the results of qRT-PCR analysis, monitoring the expressionlevels of Tmem92, Stra8, Actb (beta-actin control), and endogenousZscan4 genes after contacting MC1 mouse ES cells with Sendai virusvectors expressing either mouse Zscan4c or human ZSCAN4. Compared tocontrol cells contacted with an empty Sendai vector (SeVAG), bothSeVmZscan4 and SeVhZSCAN4 increased the expression of Tmem92, Stra8, andendogenous Zscan4 genes. SeVmZERT2 and SeVhZERT2 also function in thepresence of Tmx. Similarly, temperature-sensitive Sendai vectorsexpressing either mouse Zscan4c or human ZSCAN4 also functioned in theMCI cells (FIG. 4C).

These results indicate that Zscan4 biologics, in the form of Sendaivectors expressing Zscan4 or synthetic Zscan4 mRNAs, can function inmouse ES cells in a manner similar to the mouse Zscan4c transgeneintegrated into the mouse genome. Without wishing to be bound by theory,it is believed that Zscan4 biologics have an advantage as reagents formouse cells and therapeutics for human cells, because of the ease of useand the elimination of unwanted DNA integration into the genome.Moreover, rather than expending a great amount of time to geneticallyengineer the cells, Zscan4 biologics require only a simple procedure ofadding a Zscan4 biologic to the cell culture media.

Example 3: Effects of Zscan4 Biologics on Human Induced Pluripotent StemCells

The genes SERPINB4, DNMT3L, and DUX4 are identified as marker genes thatare upregulated when mouse Zscan4c or human ZSCAN4 gene is overexpressedby a transgene-based gene expression system.

This example demonstrates that Zscan4 biologics (e.g., expression ofZscan4, either by a synthetic mRNA encoding Zscan4 or a Sendai virusvector expressing Zscan4) can function in human iPS cells in a mannersimilar to the transgene-based Zscan4 overexpression system used inmouse pluripotent stem cells. This example also demonstrates thatsynthetic mRNAs encoding human ZSCAN4 and Sendai virus vector expressinghuman ZSCAN4 can be used as therapeutic biologics for treating humanpluripotent stem cells.

Materials and Methods Cell Culture

Human foreskin fibroblast derived-induced pluripotent stem (hiPS) cells(System Biosciences, California, USA) were cultured onmitotically-inactivated mouse embryonic fibroblasts (MEFs) and mediasupplemented with 20% knockout serum replacement and 10 ng/ml bFGF (LifeTechnologies, California, USA) following manufacturer's instructions.The medium was changed every day and cells were passaged using accutaseevery 7 days.

Synthetic mRNA

For synthesis of modified mRNA, mRNA synthesis was performed as reportedpreviously by Warren et al. (Warren et al., Cell Stem Cell, 2010 Nov. 5;7(5):618-30). Using these protocol from Warren et al., mRNAs weresynthesized by in vitro transcription of template DNAs encoding mouseZscan4c, human ZSCAN4, or green fluorescent protein (GFP) with mixturesof modified dNTPs to increase RNA stability as well as translationefficiency in mammalian cells. The following modified dNTPs were used:3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, 5-methylcytidine triphosphate,and pseudouridine triphosphate

Sendai Virus Vectors

Sendai vectors that express either mouse Zscan4c (SeV18+mZscan4/ΔF) orhuman ZSCAN4 (SeV18+hZSCAN4/ΔF) were custom-made by MBL (Medical &Biological Laboratories Co, LTD). These vectors are referred to as“SeVmZscan4” or “SeVhZSCAN4”, respectively, herein. As a control, thesame Sendai vector was used, but the vector expressed a greenfluorescent protein variant rather than Zscan4. These Sendai vectorslack the F protein, and thus, are not transmissible (Inoue et al., JVirol. 77: 23238-3246, 2003).

Sendai vectors that express either mouse Zscan4c fused to aTamoxifen-controllable ERT2 domain (SeV18+mZERT2/ΔF), or human ZSCAN4fused to Tamoxifen-controllable ERT2 domain (SeV18+hZERT2/ΔF) werecustom-made by MBL (Medical & Biological Laboratories Co, LTD). Thesevectors are referred to as “SeVmZERT2” or “SeVhZERT2”, respectively,herein. These Sendai vectors also lack the F protein, and thus, are nottransmissible (Inoue et al., J Virol. 77: 23238-3246, 2003).

Additionally, temperature-sensitive Sendai vectors that express eithermouse Zscan4 (SeV18+mZscan4/TS15ΔF) or human ZSCAN4(SeV18+hZSCAN4/TS15ΔF) were custom-made by MBL (Medical & BiologicalLaboratories Co, LTD). These vectors are referred to as“SeVmZscan4-TS15” or “SeVhZSCAN4-TS15”, respectively, herein. TheseSendai vectors are functional at 35° C., and inactive at 37° C. (Ban etal., Proc Natl Acad Sci USA. 2011; 108(34):14234-14239). As a control,the same temperature-sensitive Sendai vector was used, but the vectorexpressed a green fluorescent protein variant rather than Zscan4. Thisvector is referred to as “SeVAG-TS15” herein. These Sendai vector alsolacks the F protein, and thus, it is not transmissible (Inoue et al., JVirol. 77: 23238-3246, 2003).

Results

FIG. 5A shows that synthetic mRNAs encoding human ZSCAN4 can induce theexpression of SERPINB4. FIG. 5B shows that SeVmZscan4, SeVhZSCAN4,SeVmZERT2 (Tmx+condition), and SeVhZERT2 (Tmx+condition) can induce theexpression SERPINB4 and, to some extent, DNMT3L and DUX4. Similarly,temperature-sensitive Sendai vectors expressing either mouse Zscan4c orhuman ZSCAN4 can induce the expression of DNMT3L (FIG. 5C).

These results indicate that Zscan4 biologics, in the form of Sendaivectors expressing Zscan4 or synthetic Zscan4 mRNAs, can function inhuman pluripotent stem cells, e.g., human iPS cells, as Zscan4 inducesmarkers (e.g., SERPINB4, DNMT3L, and DUX4) for the effects of Zscan4 onhuman iPS cells. These markers may also be useful for measuring thequality and effectiveness of Zscan4 biologics (Quality Control [QC]).

Example 4: Zscan4 Expression to Improve the Quality of Human PluripotentStem Cells

This example describes the finding that Zscan4 biologics can improve thequality of human pluripotent stem cells, including but not limited tohuman embryonic stem (ES) cells and induced pluripotent stem (iPS)cells, by utilizing Zscan4 biologics (e.g., expression of Zscan4, eitherby a synthetic mRNA encoding Zscan4 or a Sendai virus vector expressingZscan4) to correct one or more chromosome abnormalities and to correctone or more epigenetic errors. In particular, it is shown that a Zscan4biologic can correct erroneous DNA methylation patterns in human iPScells by transiently demethylating several DNA regions that aredifficult to demethylate by other procedures. For example, there areregions in the genome where DNA methylation patterns are differentbetween human iPS cells and human ES cells. It is thus important tocorrect such erroneous DNA methylation patterns in human iPS cells. Itis believed that the ability of Zscan4 biologics to correct DNAmethylation problems by merely exposing human iPS cells to a Zscan4biologic may be a key technology for improving the safety of human iPScells for therapeutic uses. The ability of Zscan4 biologics to correctDNA methylation problems in human iPS cells will also allow for theimprovement of existing human iPS cells.

Example 5: Zscan4 Expression Extends the Lifespan of Human DyskeratosisCongenita Fibroblast Cells and Extends Telomere Length in HumanDyskeratosis Congenita Fibroblast Cells

This example describes the finding that expression of human ZSCAN4,either by a synthetic mRNA encoding ZSCAN4 or a Sendai virus vectorexpressing Zscan4, induces lifespan extension of human dyskeratosiscongenita fibroblast cells, and also induces telomere length elongationin human dyskeratosis congenita fibroblast cells. This example alsodemonstrates that synthetic mRNAs encoding human ZSCAN4 and Sendai virusvector expressing human ZSCAN4 can be used as therapeutic biologics.

Materials and Methods Cell Culture

Fibroblast cells isolated from a patient with dyskeratosis congenita(DKC: X-linked) were purchased from the Coriell Cell Repository (CatalogID AG04646). According to the Coriell Catalog information, the celldonor, an 11 year old male Caucasian, is affected and presented withskin eruptions, anemia, nail dystrophy and esophageal abnormalities.Family history is negative. The biopsy was taken ante-mortem fromuninvolved skin. The culture was initiated on 2/21/81 using explants ofminced tissue. The cell morphology is fibroblast-like. The populationdoubling level (PDL) was 5.41 at freeze and the passage number was 5.

After receiving the DKC cells from the Coriell Cell Repository, thecells were cultured for a few more passages. The cells were culturedunder condition recommended by the Coriell Cell Repository: Eagle'sMinimum Essential Medium with Earle's salts and non-essential aminoacids, supplemented with 15% fetal bovine serum (not inactivated).

Synthetic mRNA

For synthesis of modified mRNA, mRNA synthesis was performed as reportedpreviously by Warren et al. (Warren et al., Cell Stem Cell, 2010 Nov. 5;7(5):618-30). Using these protocol from Warren et al., mRNAs weresynthesized by in vitro transcription of template DNAs encoding humanZSCAN4 or green fluorescent protein (GFP) with mixtures of modifieddNTPs to increase RNA stability as well as translation efficiency inmammalian cells. The following modified dNTPs were used:3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, 5-methylcytidine triphosphate,and pseudouridine triphosphate.

Sendai Virus Vector

A Sendai vector that expresses human ZSCAN4 (SeV18+hZSCAN4/ΔF) wascustom-made by MBL (Medical & Biological Laboratories Co, LTD). Thevector is referred to as “SeVhZSCAN4” herein. This Sendai vector lacksthe F protein, and thus, it is non-transmissible (Inoue et al., J Virol.77:3238-3246, 2003). An MOI (multiplicity of infection) of 10 was usedfor the experiments.

Telomere Southern Blot Analysis

Telomere lengths of cells were measured by Southern blot analysis usingthe TeloTAGGG Telomere Length Assay kit (Roche Applied Sciences,Indiana, USA) according to the manufacturer's instruction.

Results Synthetic mRNAs Encoding Human ZSCAN4 Extend the Lifespan ofHuman Dyskeratosis Congenita Fibroblast Cells

DKC cells were plated in a 6 well dish at a concentration of 5×10⁴cells/well and then transfected with 1 μg of synthetic mRNAs using 5 μlof Lipofectamine (RNAiMAX: Life Technologies, California, USA). Themedium was changed the next day. Every week cells were passaged at aratio of 1:2 and transfected with synthetic mRNAs in the presence of 50ng/ml B 18R (Type I IFN inhibitor: eBiosciences, Inc., California, USA).Samples were prepared in triplicate. Cell number was counted using theAutomated Cell Counter Moxi Z (ORFLO Technologies, Idaho, USA). Cellnumbers were converted to PDL (population doubling level), starting atPDL=0 (normalized from passage 9, equivalent to approximately PDL=9according to the information from the Coriell Cell Repository).

FIG. 6 shows the results of cell growth assays over 60 days. In controlexperiments, repeated transfections of GFP mRNAs do not change thegrowth patterns of DKC cells: after approximately 20 days in culture(approximately PDL=2), cells stop proliferating and undergo cellularsenescence. By contrast, repeated transfections of human ZSCAN4 mRNAsextend the lifespan of the DKC cells and the cells continued to growuntil ˜40 days (˜PDL=4). It is important to note that the hZSCAN4treatment did not transform the cells into tumor-like cells withunlimited proliferation capacity. These results indicate that thehZSCAN4 treatment can extend the lifespan of cells from DKC patientswithout transforming the cells into tumors.

Sendai Virus Vectors Expressing Human ZSCAN4 Extend the Lifespan ofHuman Dyskeratosis Congenita Fibroblast Cells

DKC cells were plated in a 6 well dish at a concentration of 5×10⁴cells/well, and 6 hours later the cells were contacted with SeVhZSCAN4vector at an MOI of 10 for 24 hours. As a control, a second sample ofDKC cells was cultured in the same manner, but without exposure to theSeVhZSCAN4 vector. Cells were passaged every one or two weeks. Cellnumber was counted by the Automated Cell Counter Moxi Z (ORFLOTechnologies, Idaho, USA). Cell numbers were converted to PDL(population doubling level), starting at PDL=0 (normalized from passage9, equivalent to approximately PDL=9 according to the information fromthe Coriell Cell Repository).

FIG. 7A shows the results of cell growth assays. In control experiments,DKC cells show a typical growth pattern: after approximately 25 days inculture (approximately PDL=2), cells stop proliferating and undergocellular senescence. By contrast, DKC cells contacted with theSeVhZSCAN4 vector show an increase in cell lifespan. It is important tonote that the SeVhZSCAN4-treatment does not change the cells intotumor-like cells with unlimited proliferation capacity.

FIG. 7B shows cell morphologies on day 28. SeVhZSCAN4-treated cells showa better morphology than the non-treated control cells.

These results indicate that the SeVhZSCAN4 treatment can extend thelifespan of cells from DKC patients without transforming the cells intotumors.

Synthetic mRNAs Encoding Human ZSCAN4 Elongate Telomere Length in HumanDyskeratosis Congenita Fibroblast Cells

FIG. 8A shows the procedure of the experiments. DKC cells were plated in10 cm culture dishes at a concentration of 1×10⁵ cells/10 cm culturedish. Cells in each 10-cm dish were then transfected with 5 μg ofsynthetic mRNAs using 25 μl of Lipofectamine (RNAiMAX: LifeTechnologies, California, USA). The medium was changed the next day.Cells from 1 dish were harvested on day 7 and genomic DNA was extracted.Cells from another dish were passaged at a 1:2 ratio and thentransfected with 5 μg of synthetic mRNAs using 25 μl of Lipofectamine.Cells were harvested on day 15 and genomic DNA was extracted. Thegenomic DNA was then subjected to the telomere length assays.

FIG. 8B shows the results of telomere length assays. As previouslyreported (Wong J. M., Collins K. Genes Dev. (2006), 20(20), 2848-2858),telomere length of DKC fibroblast cells are shorter than that of normalcells and quickly become even shorter when the DKC cells are cultured.In control experiments, transfection with GFP-mRNAs did not change thetelomere length shortening patterns of DKC cells. By contrast,transfection with human ZSCAN4-mRNAs elongated telomere length of theDKC cells and prevented telomere lengths from getting shorter. Theseresults indicate that telomere length shortening of DKC cells, a primarycause of DKC disease phenotypes, can be rescued by treating cells withhZSCAN4-mRNAs.

Example 6: Zscan4 Expression Extends Lifespan of Human Werner SyndromeCells

WS patients are characterized by the appearance of premature aging.Cells of WS patients in culture exhibit a higher rate of chromosomalbreaks, translocations and deletions. Thus, WS patients are in need of atreatment that can enhance genome and/or chromosome stability.

This example demonstrates that synthetic mRNAs encoding Zscan4 canextend the lifespan of fibroblast cells isolated from a Werner syndrome(WS) patient. This example also demonstrates that synthetic mRNAsencoding human ZSCAN4 can be used as therapeutic biologics.

Materials and Methods Cell Culture

Fibroblast cells isolated from a patient with Werner syndrome (WS) werepurchased from the Coriell Cell Repository (Catalog ID AG03141).According to the Coriell Catalog information, “the donor was a 30 yearold female Caucasian, and had features of premature aging, pigmented andatrophic skin, cataracts and hyperlipidemia type V. The biopsy was takenante-mortem on 9/20/78. The culture was initiated using explants ofminced skin tissue. The cell morphology is fibroblast-like. Thekaryotype is 46,XX with 80% of cells examined showing random chromosomalabnormalities. Homozygous for a C to T transition at nucleotide 2476 inthe WRN gene (2476C>T), resulting in a stop codon at 748 {Gln748TER(Q748X)}. The cumulative population doubling level (PDL) was 17.97 atfreeze and the passage number was 11.” After receiving the WS cells fromthe Coriell Cell Repository, the cells were cultured for a few morepassages. The cells were cultured in the condition recommended by theCoriell Cell Repository: Eagle's Minimum Essential Medium with Earle'ssalts Earle's salts:Dulbecco's modified MEM, supplemented with 15% fetalbovine serum (not inactivated).

Synthetic mRNA

For synthesis of modified mRNA, mRNA synthesis was performed as reportedpreviously by Warren et al. (Warren et al., Cell Stem Cell, 2010 Nov. 5;7(5):618-30). Using these protocol from Warren et al., mRNAs weresynthesized by in vitro transcription of template DNAs encoding humanZSCAN4 or green fluorescent protein (GFP) with mixtures of modifieddNTPs to increase RNA stability as well as translation efficiency inmammalian cells. The following modified dNTPs were used:3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, 5-methylcytidine triphosphate,and pseudouridine triphosphate.

Results

WS cells were plated in a 6 well dish at a concentration at 5×10⁴cells/well, and then transfected with 1 μg of synthetic mRNAs using 5 μlof Lipofectamine (RNAiMAX: Life Technologies, California, USA) at day 0.The medium was changed the next day. A second transfection with the samemRNAs was carried out on day 3. Dependent on the growth of the cells,the cells were passaged at a ratio of 1:2 every 1 or 2 weeks. Sampleswere prepared in triplicate. Cell numbers were converted to PDL,starting at a PDL of 0 (approximately equivalent to a cumulative PDL of19, which is near cellular senescence according to the information fromthe Coriell Cell Repository).

FIG. 9 shows the results of cell growth assays. WS cells transfectedwith synthetic GFP mRNAs were used as a control. As shown in FIG. 9, WScells transfected with synthetic GFP mRNAs underwent cellular senescenceand stopped proliferating. By contrast, the transfection of WS cellswith synthetic hZSCAN4 mRNAs provided WS cells with two more PDL, andthus extended the lifespan of the WS cells (FIG. 9). Importantly, theresults demonstrate that synthetic hZSCAN4 mRNAs did not provideunlimited cell growth, as the cells stopped proliferating eventually.Similar to the results with synthetic hZSCAN4 mRNAs, lifespan extensionof WS cells was also observed in cells contacted with SeVhZSCAN4 orSeVhZSCAN4-TS15 Sendai virus vectors that express human ZSCAN4. Withoutwishing to be bound by theory, it is believed that the use of ZSCAN4 asa therapeutic biologic does not appear to cause cell transformationand/or cancer in treated cells. These results indicate that hZSCAN4treatment can extend the lifespan of cells in WS patients withouttransforming the cells into tumors.

Example 7: Zscan4 Expression to Treat Telomere Shortening in Patients

FIG. 10 illustrates one mode of treatment using Zscan4. This procedureis very similar to the bone marrow transplantation procedures that havebeen done routinely in hospitals to treat patients with bone marrowfailures and leukemia. Bone marrow, which includes hematopoietic stemcells and mesenchymal stem cells, will be aspirated from patients andthen immediately exposed to Zscan4 (e.g., Sendai vector carrying humanZSCAN4). This exposure to Zscan4 will be transient and for a short time.Without wishing to be bound by theory, it is believed that theexpression of Zscan4 will disappear when the bone marrow cells areadministered back into the patient. Alternatively, temperature-sensitiveSendai vectors can be used, as the expression of Zscan4 can be turnedoff by switching the temperature. Alternatively, Sendai vectors carryingthe fusion protein hZSCAN4-ERT2, which can be turned on by addingTamoxifen and turned off by removing Tamoxifen, can be used.Alternatively, synthetic mRNAs such as hZSCAN4-mRNAs can be used, as theproduction of ZSCAN4 protein is transient due to the limited half-lifeof synthetic mRNAs. The thusly Zscan4-rejuvenated bone marrow cells willthen repopulate the patient's bone marrow and hematopoietic compartment.Based on the long term effects of this transient Zscan4 contact, andwithout wishing to be bound by theory, it is believed that thisprocedure is required only once or periodically after long intervals oftime (e.g., many years). Without wishing to be bound by theory, it isbelieved that the Zscan4-rejuvenated bone marrow cells will out competeand thus replace sick bone marrow cells. Accordingly, it is believedthat irradiation of bone marrow to eliminate sick bone marrow cells,which is performed during standard bone marrow transplantation, wouldnot be necessary.

Example 8: Zscan4 Expression Extends Telomeres in Human Fibroblast Cells

This example describes the finding that Zscan4 overexpression inducestelomere extension in human fibroblast cells.

Materials and Methods Cell Culture

Primary adult human dermal fibroblasts (HDFa) isolated from adult skin(˜30 year old) were purchased from Life Technologies (Cat. no.C-013-5C). Fibroblasts (GM01309) isolated from a Fanconi anemia,complementation group A (FANCA) patient were purchased from the CoriellCell Repository. These cells were maintained under standard cultureconditions: DMEM (Dulbecco's Modified Eagle Medium) supplemented with10% Fetal Bovine Serum.

Telomere Quantification by qPCR

Telomere quantification by qPCR was conducted using procedures describedpreviously (Cawthon, R. M. Nucleic Acids Res. 2002 May 15; 30(10):e47;and Callicott, R J et al. Comparative Medicine, 2006). Genomic DNA wasextracted from >5×10⁵ fibroblast cells using the DNeasy blood and tissuekit (Qiagen). Quality of gDNA samples were assessed using Nanodrop.Genomic DNA samples with an A260/280 absorbance ratio greater than 1.8,and an A260/230 absorbance ratio of around 2 were used for qPCR todetermine telomere length.

The primers used for telomere PCR were as follows:

tel1b: (SEQ ID NO: 41) 5′-CGGTTT(GTTTGG)₅GTT-3′; and tel2b:(SEQ ID NO: 42) 5′-GGCTTG(CCTTAC)₅CCT-3′Each primer was used at a final concentration of 300 nM.

The primers used for single copy gene PCR were as follows:

36B4u: (SEQ ID NO: 43) 5′-CAGCAAGTGGGAAGGTGTAATCC-3′; and 36B4d:(SEQ ID NO: 44) 5′-CCCATTCTATCATCAACGGGTACAA-3′The 36B4u primer was used at a final concentration of 300 nM, and the36B4d primer was used at a final concentration of 500 nM.

The final 20 μl qPCR reaction was place in one well of a 96-well plateand included 20 ng gDNA, primers, and 1X Power SYBR green (AppliedBiosystems). The telomere PCR thermal cycling program for the Tellb/2bPCR was: 95° C. for 10 minutes, 40 cycles of 95° C. for 15 s, and 56° C.for 1 minute. The telomere PCR thermal cycling program for the 36B4 PCRwas: 95° C. for 10 minutes, 40 cycles of 95° C. for 15 s, and 58° C. for1 minute. The StepOne Plus qPCR machine (Applied Biosystems) was used toprocess the samples. Threshold level was set to obtain sample Ct valuesaround 20-22. The delta delta Ct method was used to calculate therelative telomere/single copy gene ratio (relative T/S ratio) forassessment of telomere length in each sample.

Sendai Virus Vectors

Sendai vectors that express human ZSCAN4 (SeV18+hZSCAN4/ΔF) werecustom-made by MBL (Medical & Biological Laboratories Co, LTD). ThisSendai vector lacks the F protein, and thus, it is non-transmissible(Inoue et al., J Virol. 77:3238-3246, 2003).

Results Overexpression of Human ZSCAN4 Rapidly Increases TelomereLengths in Normal Adult Human Fibroblast Cells

Adult human dermal fibroblasts (HDFa) were cultured under standardculture conditions. On the day after passaging cells, one sample ofcells was harvested for genomic DNA extraction (no treatment control). Asecond sample of cells was transduced with the SvhZSCAN4 Sendai viralvector.

Transduced cells were harvested 2 days (SeVhZSCAN4-treatment day 2) or 3days (SeVhZSCAN4-treatment day 3) after transduction. Telomere length ofharvested cells was then measured by qRT-PCR, and the relative telomerelength (T/S ratio) to the control no treatment control cells wascalculated.

As shown in FIG. 11, the average length of telomeres increased after day2 and after day 3. In particular, 2 days after transduction with ZSCAN4,the HDFa cells had a T/S ratio of about 1.4, while the control cells hada T/S ratio of 1.0 (FIG. 11). Similarly, 3 days after transduction withZSCAN4, the HDFa cells had a T/S ratio of about 1.4 (FIG. 11). Theseresults indicate that after two days of transduction with ZSCAN4, HDFacells had about a 40% increase in relative telomere length, as comparedto the control cells that were not transduced with ZSCAN4.

Overexpression of Human ZSCAN4 Rapidly Increases Telomere Lengths inFibroblast Isolated from a Patient with Fanconi Anemia, ComplementationGroup A

GM01309 fibroblasts isolated from a FANCA patient were cultured understandard culture conditions. On the day after passaging cells, onesample of cells was harvested for genomic DNA extraction (no treatmentcontrol). A second sample of cells was transduced with the SvhZSCAN4Sendai viral vector.

Transduced cells were harvested 2 days (SeVhZSCAN4-treatment day 2) or 3days (SeVhZSCAN4-treatment day 3) after transduction. Telomere length ofharvested cells was then measured by qRT-PCR, and the relative telomerelength (T/S ratio) to the control no treatment control cells wascalculated.

As shown in FIG. 12, the average length of telomeres increased slightlyafter day 2 and dramatically after day 3. In particular, 2 days aftertransduction with ZSCAN4, the GM01309 cells had a T/S ratio of about1.1, while the control cells had a T/S ratio of 1.0 (FIG. 12). Thisresult indicates that after two days of transduction with ZSCAN4,GM01309 cells had about a 10% increase in relative telomere length, ascompared to the control cells that were not transduced with ZSCAN4.

Three days after transduction with ZSCAN4, the GM01309 cells had a T/Sratio of about 2.6 (FIG. 12). This result indicates that after threedays of transduction with ZSCAN4, GM01309 cells had about a 160%increase in relative telomere length, as compared to the control cellsthat were not transduced with ZSCAN4.

Example 9: Zscan4 Expression Extends Lifespan of Human Fibroblast Cells

This example describes the finding that synthetic mRNAs encoding humanZSCAN4 can extend the lifespan of dermal fibroblast cells isolated froma healthy adult. This example also demonstrates that synthetic mRNAsencoding human ZSCAN4 can be used as therapeutic biologics.

Materials and Methods Cell Culture

Primary human dermal fibroblasts cells (HDFa) isolated from adult skinwere purchased from Life Technologies. According to the manufacturer'sinformation, the HDFa cells are capable of at least 12 populationdoublings (PDL). HDFa cells were cultured according to the manufacture'sinstruction. After receiving the HDFa cells, the cells were cultured formany passages such that the cells do not grow exponentially and areapproaching cellular senescence.

Synthetic mRNA

For synthesis of modified mRNA, mRNA synthesis was performed as reportedpreviously by Warren et al. (Warren et al., Cell Stem Cell, 2010 Nov. 5;7(5):618-30). Using these protocol from Warren et al., mRNAs weresynthesized by in vitro transcription of template DNAs encoding humanZSCAN4 or green fluorescent protein (GFP) with mixtures of modifieddNTPs to increase RNA stability as well as translation efficiency inmammalian cells. The following modified dNTPs were used:3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, 5-methylcytidine triphosphate,and pseudouridine triphosphate.

Results

HDFa cells at a stage near cellular senescence were plated in a 6 welldish at a concentration of 1×10⁵ cells/well, and then transfected with 1μg of synthetic mRNAs using 5 μl of Lipofectamine (RNAiMAX: LifeTechnologies, California, USA) at day 0. The medium was changed the nextday. A second transfection with the same mRNAs was carried out on day 3.Dependent on the growth of the cells, cells were passaged at a ratio of1:2 every 1 or 2 weeks. Samples were prepared in triplicate. Cellnumbers were converted to PDL, starting at a PDL of 0.

FIG. 13 shows the results of cell growth assays. HDFa cells transfectedwith synthetic GFP mRNAs were used as a control. After 2 weeks, HDFacells transfected with synthetic GFP mRNAs underwent cellular senescenceand proliferated slowly (FIG. 13). By contrast, HDFa cells transfectedwith synthetic hZSCAN4 mRNAs grew an additional 2 to 3 more PDLs, andthus, extended the lifespan of the HDFa cells near cellular senescence(FIGS. 13A and 13B). Synthetic mouse Zscan4 mRNAs did not appear toprovide lifespan extension. This result indicates that human ZSCAN4works better than mouse Zscan4c for extending the lifespan of HDFa cells(FIG. 13C). However, the synthetic hZSCAN4 mRNAs did not provideunlimited cell growth, as the cells slowed down or stopped proliferatingeventually. Similar to the results with synthetic hZSCAN4 mRNAs,lifespan extension of HDFa cells was also observed in cells contactedwith SeVhZSCAN4 or SeVhZSCAN4-TS15 Sendai virus vectors that expresshuman ZSCAN4. Thus, the use of ZSCAN4 as a therapeutic biologic was notobserved to cause cell transformation and/or cancer in treated cells.These results indicate that hZSCAN4 treatment can extend the lifespan ofHDFa cells without transforming the cells into tumors.

Example 10: Telomere Length Elongation in Human Mesenchymal Stem (MS)Cells by Zscan4 Biologics

This example describes the finding that a temperature-sensitive Sendaivirus vector that expresses human ZSCAN4 can elongate telomere length inhuman mesenchymal stem (MS) cells. This example also demonstrates thatSendai virus vectors expressing human ZSCAN4 can be used as biologics toimprove adult stem cells therapies including, without limitation, bonemarrow transplants.

Materials and Methods Cell Culture

Human adipose-derives mesenchymal stem cells (MSCs) were purchased fromLife Technologies (CA, USA). According to the manufacturer'sinformation, ADSCs have demonstrated very similar phenotypic andfunctional characteristics to bone marrow-derived mesenchymal stemcells. They can be expanded to 4-5 passages before they lose theirability to grow or differentiate into all potential phenotypes. Thecells were cultured in the condition recommended by the manufacturer.

Sendai Virus Vectors

Temperature-sensitive Sendai vectors that express either mouse Zscan4(SeV18+mZscan4/TS15ΔF) or human ZSCAN4 (SeV18+hZSCAN4/TS15ΔF) werecustom-made by MBL (Medical & Biological Laboratories Co, LTD). Thesevectors are referred to as “SeVmZscan4-TS15” or “SeVhZSCAN4-TS15”,respectively, herein. These Sendai vectors are functional at 35° C., andinactive at 37° C. (Ban et al., Proc Natl Acad Sci USA. 2011;108(34):14234-14239). As a control, the same temperature-sensitiveSendai vector was used, but the vector expressed a green fluorescentprotein variant rather than Zscan4. This vector is referred to as“SeVAG-TS15” herein. These Sendai vectors also lack the F protein, andthus, it is not transmissible (Inoue et al., J Virol. 77: 23238-3246,2003).

Telomere Southern Blot Analysis

Telomere lengths of cells were measured by Southern blot analysis usingthe TeloTAGGG Telomere Length Assay kit (Roche Applied Sciences,Indiana, USA) according to the manufacturer's instruction.

Results

Human adipose-derives mesenchymal stem cells (MSCs) at passage 2 wereplated in 10-cm dishes at a density of 1.5×10⁵ cells, and contacted withtemperature-sensitive Sendai vectors at an MOI of 10 (at day 0). Cellswere incubated in media containing 10 μM H₂O₂ and kept at 35° C. for 3days, followed by culturing at 37° C. Cells were passaged at a ratio of1:2 on day 7. After another passaging on day 10, cells were contactedagain with the temperature-sensitive Sendai vectors at an MOI of 10 andincubated 35° C. for 3 days, followed by culturing at 37° C.Subsequently, cells were passaged on day 14 (passage 3), day 20, day 27,day 42, and day 62 (passage 7). Cells were incubated in media containing10 μM H₂O₂ so that telomere lengths get shorter faster than undertypical cell culture conditions.

FIG. 14 shows the results of telomere length assays. Average telomerelengths (shown in the figure legend) were estimated based on Southernblot analysis according to manufacturer's protocol. As shown in FIG. 14,the human ZSCAN4 (SeVhZSCAN4-TS15) increased the telomere length ofhuman MSCs, whereas mouse Zscan4 (SeVmZscan4-TS) did not appear toaffect telomere length. These results indicate that telomere lengthshortening in human MSCs can be rescued by treating cells with humanZSCAN4 biologics. Without wishing to be bound by theory, it is believedthat the effects of human ZSCAN4 described herein may be applied toother human tissue stem cells in culture. It is also believed that humanZSCAN4 biologics may also elongate telomere length (e.g., cellrejuvenation) in human tissue stem cells resident in human tissue (i.e.,in vivo).

Example 11: Zscan4 Expression to Treat Patients with Defects in ResidentTissue Stem Cells

There are many diseases that are caused by one or more deficiencies inresident tissue stem cells (i.e., tissue stem cells resident in theorgan and/or tissue of the human body). For example, Duchenne musculardystrophy is known to be associated with premature aging of muscle stemcells (satellite cells). Based on the results described herein thatZscan4 biologics (e.g., expression of Zscan4, either by a synthetic mRNAencoding Zscan4 or a Sendai virus vector expressing Zscan4) canrejuvenate tissue stem cells, it is believed that Zscan4 expression inresident tissue stem cells can correct disease-associated deficienciesin the cells. In the case of Duchene muscular dystrophy, it is thoughtthat Zscan4 expression can be used to treat patients with Duchennemuscular dystrophy, by administering a Zscan4 biologic to muscle cells,particularly muscle stem cells, of a patient with Duchenne musculardystrophy, to prevent the early deterioration of muscles cells.

Example 12: Zscan4 Expression to Treat Patients with Diabetes

It has been demonstrated that human ZSCAN4 is naturally, though rarely,expressed in some tissue stem cells in human pancreas. Based on theresults described herein that Zscan4 biologics (e.g., expression ofZscan4, either by a synthetic mRNA encoding Zscan4 or a Sendai virusvector expressing Zscan4) can rejuvenate tissue stem cells andterminally differentiated cells, it is believed that ZSCAN4 expressioncan be used to treat patients with Diabetes, by administering a Zscan4biologic to pancreatic cells, particularly resident pancreatic tissuestem cells, of a patient with Diabetes, to prevent pancreatic cells fromfurther deterioration, and thereby facilitate the production ofbeta-cells.

Example 13: Zscan4 Expression to Treat Patients with Atopic Dermatitisand Other Skin Lesions

Based on the results described herein that Zscan4 biologics (e.g.,expression of Zscan4, either by a synthetic mRNA encoding Zscan4 or aSendai virus vector expressing Zscan4) can rejuvenate tissue stem cellsand terminally differentiated cells, it is believed that Zscan4expression can be used to treat patients with atopic dermatitis or otherskin lesions, by exposing a Zscan4 biologic (e.g., by topicaladministration) to the skin, particularly resident skin tissue stemcells, of a patient with atopic dermatitis or other skin lesions, toprevent the skin tissue stem cells and skin cells from furtherdeterioration, and thereby facilitate the production of new skin tissuestem cells and skin cells.

Example 14: Zscan4 Expression for Rejuvenating an Individual or forSlowing Down the Aging Process of an Individual by Rejuvenating Cells inthe Body, Including Terminally Differentiated Cells, Progenitor Cells,or Resident Tissue Stem Cells

It is thought that almost all the organs and tissues in the human bodyare maintained by resident tissue stem cells residing in the organs andtissues of the body. For example, the intestines are maintained by thecontinuous production of mature and differentiated cells from intestinalstem cells residing in the crypt of the intestine. Similarly, the skinis maintained by the continuous production of dermal epithelia from skinstem cells, whereas hairs are maintained by hair follicle stem cells.Compared to fully differentiated cells that age and deteriorate at arelatively fast pace, tissue stem cells tend to maintain their qualityand youthfulness, e.g., by maintaining telomere length (FIG. 15).However, even tissue stem cells gradually lose their youthfulness (FIG.15). Therefore, general aging and gradual loss of a youthful appearanceand function can be considered to be the result of aging anddeterioration of resident tissue stem cells.

Based on the results described herein that Zscan4 biologics (e.g.,expression of Zscan4, either by a synthetic mRNA encoding Zscan4 or aSendai virus vector expressing Zscan4) can rejuvenate embryonic stem(ES) cells and mesenchymal stem cells (MSCs), it is believed that Zscan4expression in tissue stem cells residing in each organ and/or tissue ofthe human body can be rejuvenated and thus restore the organ and/ortissue to a normal (i.e., young) appearance and function. This is basedon observations that human ZSCAN4 is naturally, though rarely, expressedin some tissue stem cells in the human pancreas. Furthermore, based onresults described herein that Zscan4 biologics (e.g., expression ofZscan4, either by a synthetic mRNA encoding Zscan4 or a Sendai virusvector expressing Zscan4) can rejuvenate terminally differentiatedcells, such as skin fibroblast cells, it is believed that Zscan4expression in terminally differentiated cells and/or progenitor cellsresiding in each organ and/or tissue of the human body can berejuvenated and thus restore the organ and/or tissue to a normal (i.e.,young) appearance and function.

This example describes a procedure for expressing Zscan4 biologics(e.g., expression of Zscan4, either by a synthetic mRNA encoding Zscan4or a Sendai virus vector expressing Zscan4) in tissue stem cells,terminally differentiated cells, and/or progenitor cells residing inorgans and tissues of the human body to rejuvenated and thus restore thebody a normal (i.e., young) appearance and function. In particular, aZscan4 biologic as described herein is administered to a subject in needthereof by either directly injecting a Zscan4 biologic to each organ andtissue of the body or injecting a Zscan4 biologic to the circulatingblood of the subject, thereby delivering the Zscan4 biologic to all theorgans and tissues in the body. Alternatively, or additionally, theZscan4 biologic may be injected into cerebrospinal fluids, therebydelivering the Zscan4 biologic to all nervous organs and tissues in thebody. Alternatively, or additionally, the Zscan4 biologic may beinjected into the lymphatic system, thereby delivering the Zscan4biologic to all lymphatic organs and tissues in the body. Alternatively,or additionally, the Zscan4 biologic may be inhaled into the lungtissue, thereby delivering the Zscan4 biologic to the lung tissue,thereby delivering the Zscan4 biologic to the lung tissue.Alternatively, or additionally, the Zscan4 biologic may be ingested,thereby delivering the Zscan4 biologic to all the digestive organs andtissues, including the esophagus, stomach, and intestines of the body.Alternatively, or additionally, the Zscan4 biologic may be injected intoportal veins, thereby delivering the Zscan4 biologic to the liver of thebody. Alternatively, or additionally, the Zscan4 biologic may betopically applied to the skin or scalp, thereby delivering the Zscan4biologic to the skin and skin appendages, such as hair follicles andsweat glands, of the body. These procedures will expose tissue stemcells, progenitor cells, and terminally differentiated cells in eachorgan and/or tissue of the subject to a Zscan4 biologic and therebyrejuvenate the tissue stem cells, progenitor cells, and/or terminallydifferentiated cells residing in the treated organ and/or tissue. It isbelieved that the overall effects of the rejuvenation of tissue stemcells, progenitor cells, and/or terminally differentiated cells in thetreated subject are the rejuvenation of the subject and/or the slowingdown of the aging process of the subject. It is also believed thatrejuvenation of tissue stem cells, progenitor cells, and/or terminallydifferentiated cells in the treated subject will result in lifespanextension of the subject.

Example 15: Zscan4 Expression Corrects Trisomy 21 in Human FibroblastCells Isolated from a Down Syndrome Patient

This example describes the finding that expression of human ZSCAN4,either by a synthetic mRNA encoding ZSCAN4 or a Sendai virus vectorexpressing ZSCAN4, can correct the trisomy 21 karyotype of fibroblastcells isolated from a Down syndrome patient. This example alsodemonstrates that synthetic mRNAs encoding human ZSCAN4 and Sendai virusvector expressing human ZSCAN4 can be used as therapeutic biologics.

Materials and Methods Cell Culture

Fibroblast cells isolated from a patient with Down syndrome (DS, trisomy21) were purchased from the Coriell Cell Repository (Catalog IDAG06872). According to the Coriell Catalog information, the donor was a1 year old female Caucasian. The donor had typical features of Downsyndrome (trisomy 21). The skin biopsy was taken post-mortem on 5/19/83.The culture was initiated using explants of minced skin tissue. Thekaryotype is 47,XX,+21. The cell morphology is fibroblast-like. Thecumulative population doubling level (PDL) was 10.5 at freeze and thepassage number was 5. After receiving the DS cells from the Coriell CellRepository, the cells were cultured for a few more passages. The cellswere cultured under the condition recommended by the Coriell CellRepository: Eagle's Minimum Essential Medium with Earle's salts andnon-essential amino acids, supplemented with 10% fetal bovine serum (notinactivated).

Synthetic mRNA

For synthesis of modified mRNA, mRNA synthesis was performed as reportedpreviously by Warren et al. (Warren et al., Cell Stem Cell, 2010 Nov. 5;7(5):618-30). Using these protocol from Warren et al., mRNAs weresynthesized by in vitro transcription of template DNAs encoding humanZSCAN4 or green fluorescent protein (GFP) with mixtures of modifieddNTPs to increase RNA stability as well as translation efficiency inmammalian cells. The following modified dNTPs were used:3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, 5-methylcytidine triphosphate,and pseudouridine triphosphate.

Sendai Virus Vectors

A Sendai vector that expresses human ZSCAN4 (SeV18+hZSCAN4/ΔF) wascustom-made by MBL (Medical & Biological Laboratories Co, LTD). Thevector is referred to as “SeVhZSCAN4” herein. This Sendai vector lacksthe F protein, and thus, it is non-transmissible (Inoue et al., J Virol.77:3238-3246, 2003). An MOI (multiplicity of infection) of 10 was usedfor the experiments.

Additionally, a temperature-sensitive Sendai vector that expresses humanZSCAN4 (SeV18+hZSCAN4/TS15ΔF) was custom-made by MBL (Medical &Biological Laboratories Co, LTD). This vector is referred to as“SeVhZSCAN4-TS15” herein. This Sendai vector is functional at 35° C.,and inactive at 37° C. (Ban et al., Proc Natl Acad Sci USA. 2011;108(34):14234-14239). This Sendai vector also lacks the F protein, andthus, it is not transmissible (Inoue et al., J Virol. 77: 23238-3246,2003). An MOI (multiplicity of infection) of 25 was used for thisexperiment.

Karyotype Analysis

In addition to regular karyotype analysis by G-banding, the copies ofchromosome 21 present in each cultured cell was counted by fluorescencein situ hybridization (FISH) using a probe that specifically hybridizesto the centromeric region of chromosome 21 (CHR21-10-GR: EmpireGenomics, New York, USA). In the interphase nucleus, the detection of 3fluorescent dots indicates trisomy 21 (Down syndrome), whereas thedetection of 2 fluorescence dots indicates normal copy number ofchromosome 21 (normal).

Results Synthetic mRNAs Encoding Human ZSCAN4 Correct ChromosomeAbnormalities in Fibroblast Cells Isolated from a Down Syndrome Patient(Trisomy 21)

Down syndrome (DS) fibroblast cells at the passage 7 were plated in a 10cm culture dish at a concentration of 5×10⁵ cells/well and thentransfected with 5 μg of synthetic mRNAs (hZSCAN4 or GFP) using 25 μl ofLipofectamine (RNAiMAX: Life Technologies, California, USA). In additionto cells transfected with GFP mRNAs, non-transfected cells were alsoused as a control. In one experiment (1× transfection), all the cellswere passaged on day 5 and then karyotypes were analyzed on day 10(passage 9). In another experiment (2× transfection), after passagingthe cells on day 5, a second transfection with either GFP mRNAs orhZSCAN4 mRNAs was carried out, and then the karyotype was analyzed onday 10 (passage 9).

FIG. 16A shows representative images of cell nuclei after carrying outFISH with chromosome 21 probes. Each dot indicates the number ofchromosome 21 present in the nucleus. Two dots indicates a normal disomyof chromosome 21, whereas three dots indicates a trisomy of chromosome21. FIG. 16B shows a summary of chromosome 21 counts in 1×transfectionexperiments: 10 days after transfecting human ZSCAN4 mRNAs, 14% of cellsnow have a normal number of Chromosome 21. FIG. 16C shows a summary ofchromosome 21 counts in 2× transfection experiments: transfecting humanZSCAN4 mRNAs twice makes 17% of cells carry a normal number ofchromosome 21. By contrast, both non-transfected cells and cellstransfected with GFP mRNAs show only a small fraction (<3%) of cellswith apparently normal chromosome 21. These are within the margin oferror. These results indicate that the introduction of human ZSCAN4mRNAs into cells can correct abnormalities in chromosome numbers.

Sendai Virus Vectors Expressing Human ZSCAN4 Correct ChromosomeAbnormalities in Fibroblast Cells Isolated from a Down Syndrome Patient(Trisomy 21)

FIG. 17A shows the experimental procedures. Down syndrome fibroblast(DS) cells were plated in a 6 well dish at a concentration of 5×10⁴cells/well at passage 8 (day 0). One day later, cells were treated withSeVhZSCAN4-TS15 at an MOI of 25 and kept at 35° C. for 3 days. Then, thedish was transferred to 37° C. and kept at 37° C. for the remainder ofthe experiment. The cells were passaged on day 9, day 12, and day 15.After passaging on day 15, cells were treated with SeVhZSCAN4 at an MOof 10. Subsequently, cells were passaged on day 18 and day 21. Afterpassaging on day 21, cells were treated with SeVhZSCAN4 at an MOI of 10.Karyotype analysis was performed on day 24 by FISH (FIG. 17B). Afterpassaging on day 30, cells were treated with SeVhZSCAN4 at an MOI of 10.After passaging on day 39, cells were treated with SeVhZSCAN4 at an MOIof 10. Karyotype analysis was performed on day 42 by FISH (FIG. 17C).Cells cultured in parallel without contacting the Sendai virus vector(no treatments) or cell cultured in parallel and contacted with thecontrol Sendai vector expressing the GFP variant were used as controls.The FISH images were scored independently by two experiencedresearchers: 2 fluorescence dots (2× chromosome 21: Normal); 3fluorescence dots (3× chromosome 21: trisomy 21, Down syndrome).

FIG. 17B shows a summary of chromosome 21 counts in the control cellsand in cells treated once with SeVhZSCAN4-TS15 and twice with SeVhZSCAN4(3× treatments). The results demonstrated that treating Down syndromefibroblast cells with the Sendai virus vector expressing human ZSCAN4induces correction of trisomy 21 in nearly 30% of the cells.

FIG. 17C shows a summary of chromosome 21 counts in untreated controlcells, in control cells treated with control Sendai vector, and in ellstreated once with SeVhZSCAN4-TS15 and four-times with SeVhZSCAN4 (5×treatments). The results indicate that Down syndrome fibroblast cellstreated repeatedly with the Sendai virus vector expressing human ZSCAN4induces correction of trisomy 21 in nearly 55% of the cells.

These results indicate that the introduction of human ZSCAN4 into cellscan correct abnormalities in chromosome numbers.

Example 16: Zscan4 Expression to Rejuvenate Oocytes and to CorrectChromosome Abnormalities in Oocytes and Preimplantation Embryos

During maternal aging, the oocyte competence to be successfullyfertilized dramatically declines and the risk of miscarriage and birthdefects increases. Recent studies have revealed that maternalage-related miscarriage and birth defects are predominantly caused bychromosome segregation errors in oocytes. At this point, there has beenno report that successfully reverse the ability of aged oocyte bypreventing or correcting chromosome segregation errors.

In normal development of mouse preimplantation embryos, endogenousZscan4 is expressed transiently and highly in 2-cell embryos (Falco etal., Dev Biol. 2007; 307: 539-50). We have also shown that the Zscan4expression is critical for normal development (Falco et al., Dev Biol.2007; 307: 539-50). Similarly, human ZSCAN4 is expressed transiently inhuman 6- to 8-cell stage embryos (Vassena et al., Development. 2011;138: 3699-709). As the zygotic genome activation occurs in 2-cell stagein mouse and in 6- to 8-cell stage in human, it is considered that theexpression of Zscan4 is required for human preimplantation embryodevelopment.

This example describes the procedure that Zscan4 biologics canrejuvenate oocytes and correct chromosomal abnormalities in mousepreimplantation embryos. Based on the functional similarity betweenmouse Zscan4 and human ZSCAN4 genes shown in this patent application,and even superiority of the human ZSCAN4 gene, it is considered that themouse embryo results can be directly applied to human embryos. Theseprocedures can be implemented in the In Vitro Fertilization (IVF)clinic. These procedures can reduce the risk of Down syndrome and otherkaryotype problems for women of all ages, which can be especiallybeneficial for those older than 35 years old, according to the pregnancyrisk guideline.

Materials and Methods Oocyte Collection

Aged mice (i.e., mice over 45 weeks of age) and young mice (8 weeks ofage) are purchased from the Jackson Laboratory. Oocytes at a germinalvesicle state (GV oocytes) are collected from the aged and young mice.Oocytes from the young mice are used as a control.

Synthetic mRNA

For synthesis of modified mRNA, mRNA synthesis is performed as reportedpreviously by Warren et al. (Warren et al., Cell Stem Cell, 2010 Nov. 5;7(5):618-30). Using the protocol from Warren et al., mRNAs aresynthesized by in vitro transcription of template DNAs encoding mouseZscan4c, human ZSCAN4, or green fluorescent protein (GFP) with mixturesof modified dNTPs to increase RNA stability as well as translationefficiency in mammalian cells. The following modified dNTPs were used:3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, 5-methylcytidine triphosphate,and pseudouridine triphosphate.

Sendai Virus Vectors

Sendai vectors that express either mouse Zscan4c (SeV18+mZscan4/ΔF) orhuman ZSCAN4 (SeV18+hZSCAN4/ΔF) are custom-made by MBL (Medical &Biological Laboratories Co, LTD). These vectors are referred to as“SeVmZscan4” or “SeVhZSCAN4”, respectively, herein. As a control, thesame Sendai vector is used, but the vector expressed a green fluorescentprotein variant rather than Zscan4. These Sendai vectors lack the Fprotein, and thus, are not transmissible (Inoue et al., J Virol. 77:23238-3246, 2003).

Sendai vectors that express either mouse Zscan4c fused to aTamoxifen-controllable ERT2 domain (SeV18+mZERT2/ΔF), or human ZSCAN4fused to Tamoxifen-controllable ERT2 domain (SeV18+hZERT2/ΔF) arecustom-made by MBL (Medical & Biological Laboratories Co, LTD). Thesevectors are referred to as “SeVmZERT2” or “SeVhZERT2”, respectively,herein. These Sendai vectors also lack the F protein, and thus, are nottransmissible (Inoue et al., J Virol. 77: 23238-3246, 2003).

Additionally, temperature-sensitive Sendai vectors that express eithermouse Zscan4 (SeV18+mZscan4/TS15ΔF) or human ZSCAN4(SeV18+hZSCAN4/TS15ΔF) are custom-made by MBL (Medical & BiologicalLaboratories Co, LTD). These vectors are referred to as“SeVmZscan4-TS15” or “SeVhZSCAN4-TS15”, respectively, herein. TheseSendai vectors are functional at 35° C., and inactive at 37° C. (Ban etal., Proc Natl Acad Sci USA. 2011; 108(34):14234-14239). As a control,the same Sendai vector is used, but the vector expressed a greenfluorescent protein variant rather than Zscan4. This vector is referredto as “SeVAG-TS15” herein. These Sendai vector also lacks the F protein,and thus, it is not transmissible (Inoue et al., J Virol. 77:23238-3246, 2003).

Results

Oocytes at a germinal vesicle state (GV oocytes) are collected andsubjected to in vitro maturation (IVM) for subsequent progression towardmeiosis I and II. During IVM, oocytes are contacted with a Zscan4biologic (e.g., either synthetic mRNAs encoding Zscan4 or Sendai virusvectors expressing Zscan4) and subsequently fertilized in vitro withsperm. Alternatively, fertilized oocytes/preimplantation embryos betweenthe one-cell (zygote) stage and the blastocyst stage are contacted witha Zscan4 biologic (e.g., either synthetic mRNAs encoding Zscan4 orSendai virus vectors expressing Zscan4). The methods of contact mayinclude standard Lipofectamine transfection of synthetic mRNAs Zscan4with, viral infection of Sendai virus vectors expressing Zscan4, andintracytoplasmic injection of a Zscan4 biologic by a micromanipulator.

The Zscan4-treated and fertilized oocytes are then cultured in KSOMculture medium for 96 hours at 37° C., 5% CO2.

Without wishing to be bound by theory it is believed thatZscan4-treatment will result in oocytes from aged mice that arecomparable to oocytes from young mice in terms of the number of zygotesthat can successfully develop to the blastocyst stage. Resultantblastocysts are transferred to a recipient female mouse to check birthrate of healthy pups. It is believed that treatment with Zscan4biologics improves the success rate of proper embryo development fromaged oocytes by improving the karyotype and quality of embryos.

Without wishing to be bound by theory it is also believed that the abovemethod can be applied to human oocytes and fertilizedoocytes/preimplantation embryos to improve the success rate of properembryo development from aged oocytes, and to improving the karyotype andquality of embryos (FIG. 18).

Example 17: Zscan4 Expression Represses Growth of Human Cancer Cells

This example describes the finding that a temperature-sensitive Sendaivirus vector expressing either mouse Zscan4 or human ZSCAN4 can repressthe proliferation of cancer cells. This example also demonstrates thatSendai virus vectors expressing Zscan4 can be used as therapeuticbiologics.

Materials and Methods Cell Culture

HCT116 human colorectal carcinoma cells were purchased from the AmericanType Culture Collection (ATCC). HCT116 cells are derived from colorectalcarcinoma of human adult male. According to ATCC, “the stem linechromosome number is near diploid with the modal number at 45 (62%) andpolyploids occurring at 6.8%. This line has a mutation in codon 13 ofthe ras proto-oncogene.” HCT116 cells were cultured according to theATCC's recommendation.

Sendai Virus Vectors

Temperature-sensitive Sendai vectors that express either mouse Zscan4(SeV18+mZscan4/TS15ΔF) or human ZSCAN4 (SeV18+hZSCAN4/TS15ΔF) werecustom-made by MBL (Medical & Biological Laboratories Co, LTD). Thesevectors are referred to as “SeVmZscan4-TS15” or “SeVhZSCAN4-TS15”,respectively, herein. These Sendai vectors are functional at 35° C., andinactive at 37° C. (Ban et al., Proc Natl Acad Sci USA. 2011;108(34):14234-14239). As a control, the same temperature-sensitiveSendai vector was used, but the vector expressed a green fluorescentprotein variant rather than Zscan4. This vector is referred to as“SeVAG-TS15” herein. These Sendai vectors lack the F protein, and thus,it is not transmissible (Inoue et al., J Virol. 77: 23238-3246, 2003).

Results

HCT116 cell (passage 9) samples were cultured at a concentration of8×10⁴ cells/well. Cell samples were treated with one of the followingSendai vectors: SeVAG-TS15 (control), SeVmZscan4-TS15, orSeVhZSCAN4-TS15 at an MOI of 20 and incubated at 35° C. (day 0). Each ofthe three treatment samples was prepared in triplicate. On day 3, eachtreatment sample was passaged and treated with the same Sendai vector.Cell number was counted by the Automated Cell Counter Moxi Z (ORFLOTechnologies, Idaho, USA). On day 7, each treatment sample was passagedand treated with the same Sendai vector. Cell number was counted (day7). On day 10, each treatment sample was passaged and treated with thesame Sendai vector. Cell numbers were counted (day 10). On day 14, eachtreatment sample was passaged and treated with the same Sendai vector.Cell numbers were counted (day 14). Cells were cultured at 35° C.throughout the experiments. Cell numbers were converted to PDL, startingat a PDL of 0 for day 0.

FIG. 19 shows the growth curve of HTC116 cell samples. Compared tocontrol groups (i.e., no treatment group and SeVAG-TS15-treated group),treatments with either SeVmZscan4-TS15 or SeVhZSCAN4-TS15 repressed theproliferation of HTC116 cells. As shown in FIG. 19, human ZSCAN4 wassurprisingly better than mouse Zscan4 in repressing cancer cell growth.Thus, these results demonstrate that human ZSCAN4 produces surprisinglysuperior results over mouse Zscan4c. These results further indicate thatthe hZSCAN4 treatment can repress the growth of cancer cells. Withoutwishing to be bound by theory, it is believed that Zscan4-expressingagents may be used for cancer therapy.

Example 18: Zscan4 Expression for Increasing DNA Repair Capacity ofHuman Cells or Individuals

This example describes the finding that Zscan4 biologics can increasethe DNA repair capacity of human cells. It is known that genotoxicagents, such as mitomycin C or cisplatin, kills human cells in adose-dependent manner. Cells exposed to a genotoxic agent and which arethen treated with a Zscan4 biologic (e.g., expression of Zscan4, eitherby a synthetic mRNA encoding Zscan4 or a Sendai virus vector expressingZscan4) become resistant to the genotoxic agent. It is found thatresistance to genotoxic agents by the Zscan4 biologic is due to theheightened capacity of DNA repair induced by Zscan4 expression in thecells. Thus, Zscan4 biologics can be used to: (1) to improve the DNArepair capacity of patients with diseases associated with a DNA repairdeficiency; (2) to protect specific tissues and/or organs, such asgonads, from being damaged by genotoxic agents, such as cancertherapeutics; and (3) to protect tissues, organs, and/or individualsfrom hazardous environments, such as the presence of toxic chemicals ornuclear fallouts.

Example 19: Repression of Zscan4 in Certain Cancer Stem Cells to TreatCancer

It is known that cancer tissues (e.g., tumors) contain cancer stemcells, which are not actively proliferating and are resistant to cancerchemotherapy (e.g., treatment with genotoxic agents such as cisplatin).It is believed that cancer stem cells can survive treatment withchemotherapy, and thus results in the recurrence of the cancer after thetreatment. As the presence of Zscan4 can provide cells with resistanceto genotoxic agents, it is believed that endogenous Zscan4 expressionoccurs in certain cancer stem cells, thus providing the cells withprotection from the genotoxic agents. As such, it is believed thatagents that reduce the expression of endogenous Zscan4, such as siRNAsor shRNAs specific for Zscan4, can be used to treat cancer stem cells ina patient with cancer to reduce or eliminate resistance to genotoxicagents in the cancer stem cells, and thus improve the patient's responseto cancer therapy.

What is claimed:
 1. A method of increasing telomere length in one ormore human cells, comprising contacting the one or more human cells withan agent that increases expression of Zscan4 in the human cell, whereinincreased expression of Zscan4 induces telomere lengthening in the oneor more human cells as compared to one or more corresponding human cellsthat are not contacted with the agent.
 2. A method of treating a subjectin need of telomere lengthening, comprising contacting one or more humancells in the subject with an agent that increases expression of Zscan4in the one or more human cells, wherein increased expression of Zscan4induces telomere lengthening in the one or more human cells.
 3. A methodof treating a subject in need of telomere lengthening, comprising: i.isolating one or more human cells in need of telomere lengthening fromthe subject; ii. contacting the one or more human cells with an agentthat increases expression of Zscan4 in the one or more human cells,wherein increasing expression of Zscan4 induces telomere lengthening inthe one or more human cells; and iii. administering the contacted one ormore human cells to the subject.
 4. A method of treating a disease orcondition associated with a telomere abnormality, comprisingadministering to a subject in need thereof an agent that increasesexpression of Zscan4 in one or more human cells in the subject, whereinincreasing expression of Zscan4 induces telomere lengthening in the oneor more human cells to treat the disease or condition associated with atelomere abnormality.
 5. A method of treating a disease or conditionassociated with a telomere abnormality, comprising: i. isolating one ormore human cells from a subject suffering from a disease or conditionassociated with a telomere abnormality; ii. contacting the one or morehuman cells with an agent that increases expression of Zscan4 in the oneor more human cells, wherein increasing expression of Zscan4 inducestelomere lengthening in the one or more human cells; and iii.administering the contacted one or more human cells to the subject totreat the disease or condition associated with a telomere abnormality.6. A method of treating a disease or condition associated with achromosome abnormality, comprising administering to a subject in needthereof an agent that increases expression of Zscan4 in one or morehuman cells in the subject, wherein increasing expression of Zscan4induces correction of the chromosome abnormality in the one or morehuman cells to treat the disease or condition associated with achromosome abnormality.
 7. A method of treating a disease or conditionassociated with a chromosome abnormality, comprising: i. isolating oneor more human cells from a subject suffering from a disease or conditionassociated with a chromosome abnormality; ii. contacting the one or morehuman cells with an agent that increases expression of Zscan4 in the oneor more human cells, wherein increasing expression of Zscan4 inducescorrection of the chromosome abnormality in the one or more human cells;and iii. administering the contacted one or more human cells to thesubject to treat the disease or condition associated with a chromosomeabnormality.
 8. A method of treating a disease or condition associatedwith a karyotype abnormality, comprising administering to a subject inneed thereof an agent that increases expression of Zscan4 in one or morehuman cells in the subject, wherein increasing expression of Zscan4induces correction of the karyotype abnormality in the one or more humancells to treat the disease or condition associated with a karyotypeabnormality.
 9. A method of treating a disease or condition associatedwith a karyotype abnormality, comprising: i. isolating one or more humancells from a subject suffering from a disease or condition associatedwith a karyotype abnormality; ii. contacting the one or more human cellswith an agent that increases expression of Zscan4 in the one or morehuman cells, wherein increasing expression of Zscan4 induces correctionof the karyotype abnormality in the one or more human cells; and iii.administering the contacted one or more human cells to the subject totreat the disease or condition associated with a karyotype abnormality.10. The method of claim 8 or claim 9, wherein the karyotype abnormalityis selected from the group consisting of a chromosome nullisomy, achromosome monosomy, a chromosome trisomy, a chromosome tetrasomy, and achromosome pentasomy.
 11. The method of claim 8 or claim 9, wherein thekaryotype abnormality is selected from the group consisting of trisomy21, trisomy 16, trisomy 18, trisomy 13, monosomy X, XXX aneuploidy, XXYaneuploidy, XYY aneuploidy, and 1p36 duplication.
 12. The method ofclaim 8 or claim 9, wherein the disease or condition associated with akaryotype abnormality is selected from the group consisting ofdup(17)(p11.2p11.2) syndrome, Pelizaeus-Merzbacher disease,dup(22)(q11.2q11.2) syndrome, cat-eye syndrome, Cri-du-chat syndrome,Wolf-Hirschhorn, Williams-Beuren syndrome, Charcot-Marie-Tooth disease,Hereditary neuropathy with liability to pressure palsies, Smith-Magenissyndrome, Neurofibromatosis, Alagille syndrome, Velocardiofacialsyndrome, DiGeorge syndrome, Steroid sulfatase deficiency, Kallmannsyndrome, Microphthalmia with linear skin defects, Adrenal hypoplasia,Glycerol kinase deficiency, Pelizaeus-Merzbacher disease,Testis-determining factor on Y, Azoospermia (factor a), Azoospermia(factor b), Azoospermia (factor c), and 1p36 deletion.
 13. The method ofany one of claims 4-12, wherein the disease or condition is one or morediseases or conditions selected from the group consisting of diseases oftelomere shortening, bone marrow failure syndromes, age-related telomereshortening diseases or disorders, and premature aging diseases ordisorders.
 14. The method of any one of claims 4-9, wherein the diseaseor condition is a disease of telomere shortening selected from the groupconsisting of dyskeratosis congenita, Hoyeraal-Hreidarsson syndrome,Revesz syndrome, Coats plus syndrome, idiopathic pulmonary fibrosis,liver cirrhosis, pancreatic fibrosis, Alzheimer's disease, andosteoarthritis.
 15. The method of any one of claims 4-9, wherein thedisease or condition is a bone marrow failure syndrome selected from thegroup consisting of Fanconi anemia, amegakaryocytic thrombocytopenia,aplastic anemia, Diamond Blackfan anemia, dyskeratosis congenita,paroxysmal nocturnal hemoglobinuria (PNH), Pearson syndrome, ShwachmanDiamond syndrome, thrombocytopenia, and myelodysplastic syndrome. 16.The method of any one of claims 4-9, wherein the disease or condition isan age-related telomere shortening disease or disorder, a prematureaging disease or disorder, or both selected from the group consisting ofWerner syndrome, Bloom's syndrome, Hutchinson-Gilford progeria syndrome,Cockayne syndrome, Xeroderma pigmentosa, Ataxia telangiectasia, RothmundThomson syndrome, Trichothiodystrophy, Juberg-Marsidi syndrome, and Downsyndrome.
 17. The method of any one of claims 4-9, wherein the diseaseor condition is one or more diseases or conditions selected from thegroup consisting of immunological deficiencies, an autoimmune disease,an autoimmune disorder, chronic ulcers, atherosclerosis, cancer, aneurologic injury, a degenerative disorder, a neurodegenerativedisorder, wound healing, muscle repair, cardiac muscle repair, cartilagereplacement, arthritis, osteoarthritis, tooth regeneration, blindness,age-related blindness due to proliferative decline of retinal pigmentedepithelial cells, deafness, bone marrow failure, bone marrow transplant,diabetes, muscular dystrophy, Duchenne muscular dystrophy, a geneticdisease, a genetic mutation, and DNA damage.
 18. The method of any oneof claims 4-9, wherein the disease or condition is a cancer selectedfrom the group consisting of cancers of the heart (e.g. angiosarcoma,fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma,fibroma, lipoma and teratoma), lung cancers (e.g., bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma);gastrointestinal tract cancers (e.g., esophagus (squamous cellcarcinoma, adenocarcinoma, leiomyosarcoma, lymphoma); stomach cancers(carcinoma, lymphoma, leiomyosarcoma); pancreatic cancers (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma); small bowel cancers (adenocarcinoma, lymphoma, carcinoidtumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma); large bowel cancers (adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma); genitourinary tract cancers (e.g.,kidney (adenocarcinoma, Wilms' tumor, nephroblastoma, lymphoma,leukemia); bladder and urethra cancers (squamous cell carcinoma,transitional cell carcinoma, adenocarcinoma); prostate cancers(adenocarcinoma, sarcoma); testis cancers (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); livercancers (e.g., hepatoma (hepatocellular carcinoma), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma); bonecancers (e.g., osteogenic sarcoma (osteosarcoma), fibrosarcoma,malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma,malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignantgiant cell tumor, chordoma, osteochondroma (osteocartilaginousexostoses), benign chondroma, chondroblastoma, chondromyxofibroma,osteoid osteoma and giant cell tumors); nervous system cancers (e.g.,skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans),meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma, pinealoma, glioblastomamultiforme, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma));gynecological cancers (e.g., uterus (endometrial carcinoma), cervix(cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovariancarcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma,endometrioid tumors, Brenner tumor, clear cell carcinoma, unclassifiedcarcinoma, granulosa-theca cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma,embryonal rhabdomyosarcoma, fallopian tubes (carcinoma)); hematologiccancers (e.g., blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma)); skin cancers(e.g., malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Kaposi's sarcoma, moles, dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis); and adrenal gland cancers (e.g.,neuroblastoma).
 19. The method of any one of claims 4-9 wherein thedisease or condition is an autoimmune disease selected from the groupconsisting of thyroiditis, Goodpasture's disease, rheumatoid arthritis,juvenile oligoarthritis, collagen-induced arthritis, adjuvant-inducedarthritis, Sjogren's syndrome, multiple sclerosis, experimentalautoimmune encephalomyelitis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, autoimmune gastric atrophy, pemphigusvulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes,myasthenia gravis, Grave's disease, Hashimoto's thyroiditis, sclerosingcholangitis, sclerosing sialadenitis, systemic lupus erythematosis,autoimmune thrombocytopenia purpura, Addison's disease, systemicsclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia,and pernicious anemia.
 20. The method of any one of claims 4-9, whereinthe disease or condition is a neurodegenerative disease selected fromthe group consisting of adrenoleukodystrophy (ALD), alcoholism,Alexander's disease, Alper's disease, Alzheimer's disease, amyotrophiclateral sclerosis, Lou Gehrig's Disease, ataxia telangiectasia, Battendisease, Spielmeyer-Vogt-Sjogren-Batten disease, bovine spongiformencephalopathy (BSE), Canavan disease, cerebral palsy, Cockaynesyndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, familialfatal insomnia, frontotemporal lobar degeneration, Huntington's disease,HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy bodydementia, neuroborreliosis, Machado-Joseph disease, Spinocerebellarataxia type 3, Multiple System Atrophy, multiple sclerosis, narcolepsy,Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease,Pick's disease, primary lateral sclerosis, prion diseases, progressivesupranuclear palsy, Refsum's disease, Sandhoff disease, Schilder'sdisease, subacute combined degeneration of spinal cord secondary toPernicious Anaemia, Spielmeyer-Vogt-Sjogren-Batten disease, Battendisease, spinocerebellar ataxia, spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, and toxicencephalopathy.
 21. A method of treating a cancer, comprisingadministering to a subject in need thereof an agent that increasesexpression of Zscan4 in one or more cancer cells in the subject, whereinincreasing expression of Zscan4 represses growth of the one or morecancer cells, thereby treating the cancer.
 22. A method of improvingresponsiveness to chemotherapy in a cancer patient, comprisingadministering to a subject in need thereof an agent that reducesexpression of endogenous ZSCAN4 in one or more cancer stem cells in thesubject, wherein reducing expression of endogenous ZSCAN4 reduces oreliminates resistance to one or more chemotherapeutic agents in the oneor more cancer stem cells, thereby improving responsiveness to the oneor more chemotherapeutic agents in the subject.
 23. The method of claim22, wherein the agent that reduces expression of endogenous ZSCAN4 is ansiRNA or shRNA specific for ZSCAN4.
 24. The method of any one of claims21-23, wherein the cancer selected from the group consisting of cancersof the heart (e.g. angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma), lungcancers (e.g., bronchogenic carcinoma (squamous cell, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hamartoma, mesothelioma); gastrointestinal tract cancers(e.g., esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma); stomach cancers (carcinoma, lymphoma,leiomyosarcoma); pancreatic cancers (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma); small bowel cancers(adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma); large bowelcancers (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); genitourinary tract cancers (e.g., kidney (adenocarcinoma,Wilms' tumor, nephroblastoma, lymphoma, leukemia); bladder and urethracancers (squamous cell carcinoma, transitional cell carcinoma,adenocarcinoma); prostate cancers (adenocarcinoma, sarcoma); testiscancers (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma); liver cancers (e.g., hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma); bone cancers (e.g.,osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor,chordoma, osteochondroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors); nervous system cancers (e.g., skull (osteoma,hemangioma, granuloma, xanthoma, osteitis deformans), meninges(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,medulloblastoma, glioma, ependymoma, germinoma, pinealoma, glioblastomamultiforme, oligodendroglioma, schwannoma, retinoblastoma, congenitaltumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma));gynecological cancers (e.g., uterus (endometrial carcinoma), cervix(cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovariancarcinoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma,endometrioid tumors, Brenner tumor, clear cell carcinoma, unclassifiedcarcinoma, granulosa-theca cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma,embryonal rhabdomyosarcoma, fallopian tubes (carcinoma)); hematologiccancers (e.g., blood (myeloid leukemia (acute and chronic), acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma (malignant lymphoma)); skin cancers(e.g., malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Kaposi's sarcoma, moles, dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis); and adrenal gland cancers (e.g.,neuroblastoma).
 25. A method of increasing genome stability of one ormore human cells, comprising contacting the one or more human cells withan agent that increases expression of Zscan4 in the one or more humancells, wherein increased expression of Zscan4 increases genome stabilityin the one or more human cells as compared to one or more correspondinghuman cells that are not contacted with the agent.
 26. A method ofincreasing DNA repair capacity of one or more human cells, comprisingcontacting the one or more human cells with an agent that increasesexpression of Zscan4 in the one or more human cells, wherein increasedexpression of Zscan4 increases DNA repair capacity in the one or morehuman cells as compared to one or more corresponding human cells thatare not contacted with the agent.
 27. A method of rejuvenating one ormore human cells, comprising contacting the one or more human cells withan agent that increases expression of Zscan4 in the one or more humancells, wherein increased expression of Zscan4 rejuvenates the one ormore human cells as compared to one or more corresponding human cellsthat are not contacted with the agent.
 28. A method of rejuvenatingskin, treating atopic dermatitis, and/or a skin lesion, comprisingtopically administering to the skin of a subject in need thereof anagent that increases expression of Zscan4.
 29. A method of treating hairloss, comprising topically administering to the scalp of a subject inneed thereof an agent that increases expression of Zscan4.
 30. A methodof preventing hair graying, treating hair graying, or both, comprisingadministering to one or more hair follicles of a subject in need thereofan agent that increases expression of Zscan4.
 31. A method ofrejuvenating a cornea, comprising administering to a cornea of a subjectin need thereof an agent that increases expression of Zscan4.
 32. Amethod of treating dry eye, comprising administering to a cornea of asubject in need thereof an agent that increases expression of Zscan4.33. A method of treating idiopathic pulmonary fibrosis, comprisingadministering to a lung of a subject in need thereof an agent thatincreases expression of Zscan4.
 34. A method of treatingatherosclerosis, a coronary heart disease, or both, comprisingadministering to the bloodstream of a subject in need thereof an agentthat increases expression of Zscan4.
 35. A method of providingresistance to one or more genotoxic agents in one or more human cells,comprising contacting the one or more human cells with an agent thatincreases expression of Zscan4 in the one or more human cells, whereinincreased expression of Zscan4 increases resistance to one or moregenotoxic agents in the one or more human cells as compared to one ormore corresponding human cells that are not contacted with the agent.36. The method of claim 35, wherein the genotoxic agent is mitomycin Cor cisplatin.
 37. The method of any one of claims 1-36, wherein the oneor more human cells are human adult cells.
 38. The method of any one ofclaims 1-36, wherein the one or more human cells are adult stem cells,tissue stem cells, progenitor cells, or induced pluripotent stem cells.39. The method of any one of claims 1-36, wherein the one or more humancells are one or more adult stem cells, tissue stem cells, or progenitorcells selected from the group consisting of hematopoietic stem cells,mesenchymal stem cells, adipose stem cells, neuronal stem cells, andgerm stem cells.
 40. The method of any one of claims 1-36, wherein theone or more human cells are somatic cells, mature cells, ordifferentiated cells.
 41. The method of any one of claims 1-36, whereinthe one or more human cells are one or more somatic cells, mature cells,or differentiated cells selected from the group consisting of epidermalcells, fibroblasts, lymphocytes, hepatocytes, epithelial cells,myocytes, chondrocytes, osteocytes, adipocytes, cardiomyocytes,pancreatic (3 cells, keratinocytes, erythrocytes, peripheral bloodcells, neurocytes, astrocytes, germ cells, sperm cells, and oocytes. 42.A method for inducing a human embryonic stem cell-like DNA methylationpattern in one or more human induced pluripotent stem (iPS) cells,comprising contacting the one or more human iPS cells with an agent thatincreases expression of Zscan4 in the one or more human iPS cells,wherein increased expression of Zscan4 induces a human embryonic stemcell-like DNA methylation pattern in the one or more human iPS cells ascompared to one or more corresponding human iPS cells that are notcontacted with the agent.
 43. A method of rejuvenating one or more humanoocyte cells, comprising contacting the one or more human oocyte cellswith an agent that increases expression of Zscan4 in the one or morehuman oocyte cells, wherein increased expression of Zscan4 rejuvenatesthe one or more human oocyte cells as compared to one or morecorresponding human oocyte cells that are not contacted with the agent.44. A method of increasing genome stability of one or more human oocytecells, comprising contacting the one or more human oocyte cells with anagent that increases expression of Zscan4 in the one or more humanoocyte cells, wherein increased expression of Zscan4 increases genomestability in the one or more human oocyte cells as compared to one ormore corresponding human oocyte cells that are not contacted with theagent.
 45. A method of correcting one or more karyotype abnormalities inone or more human oocyte cells, comprising contacting the one or morehuman oocyte cells with an agent that increases expression of Zscan4 inthe one or more human oocyte cells, wherein increased expression ofZscan4 induces correction of the one or more karyotype abnormalities inthe one or more human oocyte cells as compared to one or morecorresponding human oocyte cells that are not contacted with the agent.46. The method of any one of claims 43-45, wherein the one or more humanoocyte cells are isolated from a subject prior to contacting with theagent that increases expression of Zscan4.
 47. The method of any one ofclaims 43-46, wherein after contacting with the agent that increasesexpression of Zscan4 the one or more human oocyte cells undergo in vitrofertilization.
 48. An in vitro method of increasing genome stability ofone or more fertilized human oocytes, comprising contacting the one ormore fertilized human oocytes with an agent that increases expression ofZscan4 in the one or more fertilized human oocytes, wherein increasedexpression of Zscan4 increases genome stability in the one or morefertilized human oocytes as compared to one or more correspondingfertilized human oocytes that are not contacted with the agent.
 49. Anin vitro method of correcting one or more karyotype abnormalities in oneor more fertilized human oocytes, comprising contacting the one or morefertilized human oocytes with an agent that increases expression ofZscan4 in the one or more fertilized human oocytes, wherein increasedexpression of Zscan4 induces correction of the one or more karyotypeabnormalities in the one or more fertilized human oocytes as compared toone or more corresponding fertilized human oocytes that are notcontacted with the agent.
 50. The method of claim 48 or claim 49,wherein the one or more fertilized human oocytes were fertilized by invitro fertilization.
 51. The method of claim 50, wherein prior to beingfertilized, the one or more human oocytes were isolated from a subject.52. The method of any one of claims 48-51, wherein the one or morefertilized oocytes are embryos between the one-cell stage and theblastocyst stage.
 53. A method of treating a disease or conditionassociated with a telomere abnormality, comprising: i. isolating humanbone marrow cells from a subject suffering from a disease or conditionassociated with a telomere abnormality; ii. contacting the human bonemarrow cells with an agent that increases expression of Zscan4 in thehuman bone marrow cells, wherein increasing expression of Zscan4 inducestelomere lengthening in the human bone marrow cells; and iii. engraftingthe contacted human bone marrow cells into the subject to treat thedisease or condition associated with a telomere abnormality.
 54. Amethod of treating a disease or condition associated with a chromosomeabnormality, comprising: i. isolating human bone marrow cells from asubject suffering from a disease or condition associated with achromosome abnormality; ii. contacting the human bone marrow cells withan agent that increases expression of Zscan4 in the human bone marrowcells, wherein increasing expression of Zscan4 induces correction of thechromosome abnormality in the human bone marrow cells; and iii.engrafting the contacted human bone marrow cells into the subject totreat the disease or condition associated with a chromosome abnormality.55. The method of claim 53 or claim 54, wherein the disease or conditionis one or more diseases or conditions selected from the group consistingof diseases of telomere shortening, bone marrow failure syndromes,age-related telomere shortening diseases or disorders, and prematureaging diseases or disorders.
 56. The method of claim 53 or claim 54,wherein the disease or condition is a disease of telomere shorteningselected from the group consisting of dyskeratosis congenita,Hoyeraal-Hreidarsson syndrome, Revesz syndrome, Coats plus syndrome,idiopathic pulmonary fibrosis, liver cirrhosis, pancreatic fibrosis,Alzheimer's disease, and osteoarthritis.
 57. The method of claim 53 orclaim 54, wherein the disease or condition is a bone marrow failuresyndrome selected from the group consisting of Fanconi anemia,amegakaryocytic thrombocytopenia, aplastic anemia, Diamond Blackfananemia, dyskeratosis congenita, paroxysmal nocturnal hemoglobinuria(PNH), Pearson syndrome, Shwachman Diamond syndrome, thrombocytopenia,and myelodysplastic syndrome.
 58. The method of claim 53 or claim 54,wherein the disease or condition is an age-related telomere shorteningdisease or disease, a premature aging disease or disease, or bothselected from the group consisting of Werner syndrome, Bloom's syndrome,Hutchinson-Gilford progeria syndrome, Cockayne syndrome, Xerodermapigmentosa, Ataxia telangiectasia, Rothmund Thomson syndrome,Trichothiodystrophy, Juberg-Marsidi syndrome, and Down syndrome.
 59. Amethod of rejuvenating a tissue or organ in a subject, comprisingadministering to a subject in need thereof an agent that increasesexpression of Zscan4 in the tissue or organ, wherein increasingexpression of Zscan4 rejuvenates the tissue or organ.
 60. A method ofrejuvenating a subject in need thereof, comprising administering to thesubject an agent that increases expression of Zscan4, wherein increasingexpression of Zscan4 rejuvenates the subject.
 61. A method of extendinglifespan of one or more human cells, comprising contacting the one ormore human cells with an agent that increases expression of Zscan4 inone or more human cells in the subject, wherein increasing expression ofZscan4 extends the lifespan of the one or more human cells as comparedto one or more corresponding human cells that are not contacted with theagent.
 62. A method of extending lifespan of a tissue or organ in asubject, comprising administering to a subject in need thereof an agentthat increases expression of Zscan4 in the tissue or organ, whereinincreasing expression of Zscan4 extends the lifespan of the tissue ororgan.
 63. A method of extending lifespan of a subject, comprisingadministering to a subject in need thereof an agent that increasesexpression of Zscan4 in one or more human cells in the subject, whereinincreasing expression of Zscan4 extends the lifespan of the one or morehuman cells, thereby extending the lifespan of the subject.
 64. A methodof extending lifespan of a subject in need thereof, comprising: i.isolating one or more human cells from the subject; ii. contacting theone or more human cells with an agent that increases expression ofZscan4 in the one or more human cells, wherein increasing expression ofZscan4 extends the lifespan of the one or more human cells; and iii.administering the contacted one or more human cells to the subject toextend the lifespan of the subject.
 65. A method for determining one ormore Zscan4-induced effects in one or more human cells, comprising: i.contacting the one or more human cells with an agent that increasesexpression of Zscan4 in one or more human cells; ii. measuringexpression levels of SERPINB4, DNMT3L, and/or DUX4 in the one or morehuman cells; and iii. comparing the expression levels of SERPINB4,DNMT3L, and/or DUX4 in the one or more human cells to the expressionlevels of SERPINB4, DNMT3L, and/or DUX4 in one or more correspondinghuman cells that are not contacted with the agent, wherein an increasein the expression levels of SERPINB4, DNMT3L, and/or DUX4 in the one ormore human cells indicates the presence of one or more Zscan4-inducedeffects in the one or more human cell.
 66. The method of any one ofclaims 1-65, wherein the increased expression of Zscan4 is transient.67. The method of any one of claims 1-66, wherein the agent increasesZscan4 expression for about 1 hour to about 23 hours.
 68. The method ofany one of claims 1-66, wherein the agent increases Zscan4 expressionfor about 1 day to about 10 days.
 69. The method of any one of claims1-68, wherein the agent interacts directly with endogenous Zscan4 toincrease expression of Zscan4.
 70. The method of any one of claims 1-68,wherein the agent is an isolated nucleic acid molecule encoding Zscan4.71. The method of claim 70, wherein the isolated nucleic acid moleculeis a synthetic mRNA.
 72. The method of claim 70, wherein the isolatednucleic acid molecule comprises a vector.
 73. The method of claim 72,wherein the vector is a viral vector.
 74. The method of claim 73,wherein the viral vector is a paramyxovirus vector, a retrovirus vector,a lentivirus vector, or an adenovirus vector.
 75. The method of claim73, wherein the viral vector is a paramyxovirus vector.
 76. The methodof claim 75, wherein the paramyxovirus vector is a Sendai virus vector.77. The method of claim 72, wherein the vector is a plasmid vector. 78.The method of any one of claims 72-77, wherein the vector encodes Zscan4operably linked to a promoter.
 79. The method of claim 78, wherein thepromoter is a constitutive promoter.
 80. The method of claim 78, whereinthe promoter is an inducible promoter.
 81. The method of any one ofclaims 70-80, wherein the Zscan4 is a Zscan4-ERT2 fusion protein. 82.The method of any one of claims 70-80, wherein the Zscan4 is a Zscan4-ΔCprotein.
 83. The method of claim 82, wherein the Zscan4-ΔC proteincomprises a deletion of at least one zinc finger domain.
 84. The methodof any one of claims 70-83, wherein the Zscan4 is mouse Zscan4, humanZSCAN4, or a homolog thereof.
 85. The method of any one of claims 70-83,wherein the Zscan4 is selected from the group consisting of Zscan4a,Zscan4b, Zscan4c, Zscan4d, Zscan4e, and Zscan4f.
 86. The method of anyone of claims 70-83, wherein the isolated nucleic acid moleculecomprises a nucleotide sequence that is at least 70%, at least 75%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to a nucleotide sequence selected from thegroup consisting of SEQ ID Nos: 1-10 and 21-30.
 87. The method of anyone of claims 70-83, wherein the Zscan4 is human ZSCAN4.
 88. The methodof any one of claims 70-83, wherein the isolated nucleic acid moleculecomprises a nucleotide sequence that is at least 70%, at least 75%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to SEQ ID NO:
 7. 89. The method of any oneof claims 1-68, wherein the agent is a Zscan4 protein.
 90. The method ofclaim 89, wherein the Zscan4 protein is fused to a cell-penetratingpeptide.
 91. The method of claim 90, wherein the cell-penetratingpeptide comprises a protein transduction domain.
 92. The method of claim90 or claim 91, wherein the cell-penetrating peptide comprises apoly-arginine peptide tag.
 93. The method of claim 89, wherein theZscan4 protein is encapsulated in a nanoparticle.
 94. The method of anyone of claims 89-93, wherein the Zscan4 protein is a mouse Zscan4protein, a human ZSCAN4 protein, or a homolog thereof.
 95. The method ofany one of claims 89-93, wherein the Zscan4 protein is selected from thegroup consisting of a Zscan4a protein, a Zscan4b protein, a Zscan4cprotein, a Zscan4d protein, a Zscan4e protein, and a Zscan4f protein.96. The method of any one of claims 89-93, wherein the Zscan4 proteincomprises an amino acid sequence that is at least 70%, at least 75%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 11-20 and 31-40.
 97. The method of anyone of claims 89-93, wherein the Zscan4 protein is a human ZSCAN4protein.
 98. The method of any one of claims 89-93, wherein the Zscan4protein comprises an amino acid sequence that is at least 70%, at least75%, at least 80%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical to SEQ ID NO:
 17. 99. The method ofany one of claims 89-98, wherein the Zscan4 protein is a Zscan4-ERT2fusion protein.
 100. The method of any one of claims 89-93, wherein theZscan4 protein is a Zscan4-ΔC protein.
 101. The method of claim 100,wherein the Zscan4-ΔC protein comprises a mouse Zscan4 protein, a humanZSCAN4 protein, or a homolog thereof, and wherein the Zscan4 proteincomprises a deletion of at least one zinc finger domain.
 102. The methodof claim 100, wherein the Zscan4-ΔC protein comprises a Zscan4 proteinselected from the group consisting of a Zscan4a protein, a Zscan4bprotein, a Zscan4c protein, a Zscan4d protein, a Zscan4e protein, and aZscan4f protein, and wherein the Zscan4 protein comprises a deletion ofat least one zinc finger domain.
 103. The method of claim 100, whereinthe Zscan4-ΔC protein comprises a human ZSCAN4 protein, and wherein theZSCAN4 protein comprises a deletion of at least one zinc finger domain.104. The method of any one of claims 1-68, wherein the agent is aretinoid or an agent that induces oxidative stress.